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Preexisting anti-interferon-α (anti-IFN-α) autoantibodies in blood are associated with susceptibility to life-threatening COVID-19. However, it is unclear whether anti-IFN-α autoantibodies in the airways, the initial site of infection, can also determine disease outcomes. In this study, we developed a multiparameter technology, FlowBEAT, to quantify and profile the isotypes of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and anti-IFN-α antibodies in longitudinal samples collected over 20 months from the airways and blood of 129 donors spanning mild to severe COVID-19. We found that nasal IgA1 anti-IFN-α autoantibodies were induced after infection onset in more than 70% of mild and moderate COVID-19 cases and were associated with robust anti-SARS-CoV-2 immunity, fewer symptoms, and efficient recovery. Nasal anti-IFN-α autoantibodies followed the peak of host IFN-α production and waned with disease recovery, revealing a regulated balance between IFN-α and anti-IFN-α response. In contrast, systemic IgG1 anti-IFN-α autoantibodies appeared later and were detected only in a subset of patients with elevated systemic inflammation and worsening symptoms. These data reveal a protective role for nasal anti-IFN-α in the immunopathology of COVID-19 and suggest that anti-IFN-α autoantibodies may serve a homeostatic function to regulate host IFN-α after viral infection in the respiratory mucosa.

Many infections, including malaria, are associated with an increase in autoantibodies (AAbs). Prior studies have reported an association between genetic markers of susceptibility to autoimmune disease and resistance to malaria, but the underlying mechanisms are unclear. Here, we performed a longitudinal study of children and adults (n = 602) in Mali and found that high levels of plasma AAbs before the malaria season independently predicted a reduced risk of clinical malaria in children during the ensuing malaria season. Baseline AAb seroprevalence increased with age and asymptomatic Plasmodium falciparum infection. We found that AAbs purified from the plasma of protected individuals inhibit the growth of blood-stage parasites and bind P. falciparum proteins that mediate parasite invasion. Protected individuals had higher plasma immunoglobulin G (IgG) reactivity against 33 of the 123 antigens assessed in an autoantigen microarray. This study provides evidence in support of the hypothesis that a propensity toward autoimmunity offers a survival advantage against malaria., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple autoantibody types, some of which are produced by long-lived plasma cells (LLPC). Active SLE generates increased circulating antibody-secreting cells (ASC). Here, we examine the phenotypic, molecular, structural, and functional features of ASC in SLE. Relative to post-vaccination ASC in healthy controls, circulating blood ASC from patients with active SLE are enriched with newly generated mature CD19 - CD138 + ASC, similar to bone marrow LLPC. ASC from patients with SLE displayed morphological features of premature maturation and a transcriptome epigenetically initiated in SLE B cells. ASC from patients with SLE exhibited elevated protein levels of CXCR4, CXCR3 and CD138, along with molecular programs that promote survival. Furthermore, they demonstrate autocrine production of APRIL and IL-10, which contributed to their prolonged in vitro survival. Our work provides insight into the mechanisms of generation, expansion, maturation and survival of SLE ASC., (© 2024. The Author(s).)

While immunologic correlates of COVID-19 have been widely reported, their associations with post-acute sequelae of COVID-19 (PASC) remain less clear. Due to the wide array of PASC presentations, understanding if specific disease features associate with discrete immune processes and therapeutic opportunities is important. Here we profile patients in the recovery phase of COVID-19 via proteomics screening and machine learning to find signatures of ongoing antiviral B cell development, immune-mediated fibrosis, and markers of cell death in PASC patients but not in controls with uncomplicated recovery. Plasma and immune cell profiling further allow the stratification of PASC into inflammatory and non-inflammatory types. Inflammatory PASC, identifiable through a refined set of 12 blood markers, displays evidence of ongoing neutrophil activity, B cell memory alterations, and building autoreactivity more than a year post COVID-19. Our work thus helps refine PASC categorization to aid in both therapeutic targeting and epidemiological investigation of PASC., (© 2023. The Author(s).)

Severe SARS-CoV-2 infection 1 has been associated with highly inflammatory immune activation since the earliest days of the COVID-19 pandemic 2-5 . More recently, these responses have been associated with the emergence of self-reactive antibodies with pathologic potential 6-10 , although their origins and resolution have remained unclear 11 . Previously, we and others have identified extrafollicular B cell activation, a pathway associated with the formation of new autoreactive antibodies in chronic autoimmunity 12,13 , as a dominant feature of severe and critical COVID-19 (refs. 14-18 ). Here, using single-cell B cell repertoire analysis of patients with mild and severe disease, we identify the expansion of a naive-derived, low-mutation IgG1 population of antibody-secreting cells (ASCs) reflecting features of low selective pressure. These features correlate with progressive, broad, clinically relevant autoreactivity, particularly directed against nuclear antigens and carbamylated proteins, emerging 10-15 days after the onset of symptoms. Detailed analysis of the low-selection compartment shows a high frequency of clonotypes specific for both SARS-CoV-2 and autoantigens, including pathogenic autoantibodies against the glomerular basement membrane. We further identify the contraction of this pathway on recovery, re-establishment of tolerance standards and concomitant loss of acute-derived ASCs irrespective of antigen specificity. However, serological autoreactivity persists in a subset of patients with postacute sequelae, raising important questions as to the contribution of emerging autoreactivity to continuing symptomology on recovery. In summary, this study demonstrates the origins, breadth and resolution of autoreactivity in severe COVID-19, with implications for early intervention and the treatment of patients with post-COVID sequelae., (© 2022. The Author(s).)

Objective: While the contribution of B-cells to SLE is well established, its role in chronic cutaneous lupus erythematosus (CCLE) remains unclear. Here, we compare B-cell and serum auto-antibody profiles between patients with systemic lupus erythematosus (SLE), CCLE, and overlap conditions., Methods: B-cells were compared by flow cytometry amongst healthy controls, CCLE without systemic lupus (CCLE+/SLE-) and SLE patients with (SLE+/CCLE+) or without CCLE (SLE+/CCLE-). Serum was analyed for autoreactive 9G4+, anti-double-stranded DNA, anti-chromatin and anti-RNA antibodies by ELISA and for anti-RNA binding proteins (RBP) by luciferase immunoprecipitation., Results: Patients with CCLE+/SLE- share B-cell abnormalities with SLE including decreased unswitched memory and increased effector B-cells albeit at a lower level than SLE patients. Similarly, both SLE and CCLE+/SLE- patients have elevated 9G4+ IgG autoantibodies despite lower levels of anti-nucleic acid and anti-RBP antibodies in CCLE+/SLE-. CCLE+/SLE- patients could be stratified into those with SLE-like B-cell profiles and a separate group with normal B-cell profiles. The former group was more serologically active and more likely to have disseminated skin lesions., Conclusion: CCLE displays perturbations in B-cell homeostasis and partial B-cell tolerance breakdown. Our study demonstrates that this entity is immunologically heterogeneous and includes a disease segment whose B-cell compartment resembles SLE and is clinically associated with enhanced serological activity and more extensive skin disease. This picture suggests that SLE-like B-cell changes in primary CCLE may help identify patients at risk for subsequent development of SLE. B-cell profiling in CCLE might also indentify candidates who would benefit from B-cell targeted therapies., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

A wide spectrum of clinical manifestations has become a hallmark of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) COVID-19 pandemic, although the immunological underpinnings of diverse disease outcomes remain to be defined. We performed detailed characterization of B cell responses through high-dimensional flow cytometry to reveal substantial heterogeneity in both effector and immature populations. More notably, critically ill patients displayed hallmarks of extrafollicular B cell activation and shared B cell repertoire features previously described in autoimmune settings. Extrafollicular activation correlated strongly with large antibody-secreting cell expansion and early production of high concentrations of SARS-CoV-2-specific neutralizing antibodies. Yet, these patients had severe disease with elevated inflammatory biomarkers, multiorgan failure and death. Overall, these findings strongly suggest a pathogenic role for immune activation in subsets of patients with COVID-19. Our study provides further evidence that targeted immunomodulatory therapy may be beneficial in specific patient subpopulations and can be informed by careful immune profiling.

The maintenance of immunological tolerance of B lymphocytes is a complex and critical process that must be implemented as to avoid the detrimental development of autoreactivity and possible autoimmunity. Murine models have been invaluable to elucidate many of the key components in B-cell tolerance; however, translation to human homeostatic and pathogenic immune states can be difficult to assess. Functional autoreactive, flow cytometric, and single-cell cloning assays have proven to be critical in deciphering breaks in B-cell tolerance within autoimmunity; however, newer approaches to assess human B-cell tolerance may prove to be vital in the further exploration of underlying tolerance defects. In this review, we supply a comprehensive overview of human immune tolerance checkpoints with associated mechanisms of enforcement, and highlight current and future methodologies which are likely to benefit future studies into the mechanisms that become defective in human autoimmune conditions., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The increasingly recognized role of different types of B cells and plasma cells in protective and pathogenic immune responses combined with technological advances have generated a plethora of information regarding the heterogeneity of this human immune compartment. Unfortunately, the lack of a consistent classification of human B cells also creates significant imprecision on the adjudication of different phenotypes to well-defined populations. Additional confusion in the field stems from: the use of non-discriminatory, overlapping markers to define some populations, the extrapolation of mouse concepts to humans, and the assignation of functional significance to populations often defined by insufficient surface markers. In this review, we shall discuss the current understanding of human B cell heterogeneity and define major parental populations and associated subsets while discussing their functional significance. We shall also identify current challenges and opportunities. It stands to reason that a unified approach will not only permit comparison of separate studies but also improve our ability to define deviations from normative values and to create a clean framework for the identification, functional significance, and disease association with new populations., (Copyright © 2019 Sanz, Wei, Jenks, Cashman, Tipton, Woodruff, Hom and Lee.)

Systemic lupus erythematosus (SLE) is characterized by the expansion of extrafollicular pathogenic B cells derived from newly activated naive cells. Although these cells express distinct markers, their epigenetic architecture and how it contributes to SLE remain poorly understood. To address this, we determined the DNA methylomes, chromatin accessibility profiles and transcriptomes from five human B cell subsets, including a newly defined effector B cell subset, from subjects with SLE and healthy controls. Our data define a differentiation hierarchy for the subsets and elucidate the epigenetic and transcriptional differences between effector and memory B cells. Importantly, an SLE molecular signature was already established in resting naive cells and was dominated by enrichment of accessible chromatin in motifs for AP-1 and EGR transcription factors. Together, these factors acted in synergy with T-BET to shape the epigenome of expanded SLE effector B cell subsets. Thus, our data define the molecular foundation of pathogenic B cell dysfunction in SLE.

Although B cells expressing the IFNγR or the IFNγ-inducible transcription factor T-bet promote autoimmunity in Systemic Lupus Erythematosus (SLE)-prone mouse models, the role for IFNγ signaling in human antibody responses is unknown. We show that elevated levels of IFNγ in SLE patients correlate with expansion of the T-bet expressing IgD neg CD27 neg CD11c + CXCR5 neg (DN2) pre-antibody secreting cell (pre-ASC) subset. We demonstrate that naïve B cells form T-bet hi pre-ASCs following stimulation with either Th1 cells or with IFNγ, IL-2, anti-Ig and TLR7/8 ligand and that IL-21 dependent ASC formation is significantly enhanced by IFNγ or IFNγ-producing T cells. IFNγ promotes ASC development by synergizing with IL-2 and TLR7/8 ligands to induce genome-wide epigenetic reprogramming of B cells, which results in increased chromatin accessibility surrounding IRF4 and BLIMP1 binding motifs and epigenetic remodeling of IL21R and PRDM1 loci. Finally, we show that IFNγ signals poise B cells to differentiate by increasing their responsiveness to IL-21., Competing Interests: EZ, SS, CS, SJ, AN, BM, AR, DB, JB, WW, TP, MD, JE, SB, RK, WC, TS, AR, JB, IS, FL No competing interests declared, (© 2019, Zumaquero et al.)

Chronic autoimmune diseases, and in particular Systemic Lupus Erythematosus (SLE), are endowed with a long-standing autoreactive B-cell compartment that is presumed to reactivate periodically leading to the generation of new bursts of pathogenic antibody-secreting cells (ASC). Moreover, pathogenic autoantibodies are typically characterized by a high load of somatic hypermutation and in some cases are highly stable even in the context of prolonged B-cell depletion. Long-lived, highly mutated antibodies are typically generated through T-cell-dependent germinal center (GC) reactions. Accordingly, an important role for GC reactions in the generation of pathogenic autoreactivity has been postulated in SLE. Nevertheless, pathogenic autoantibodies and autoimmune disease can be generated through B-cell extrafollicular (EF) reactions in multiple mouse models and human SLE flares are characterized by the expansion of naive-derived activated effector B cells of extrafollicular phenotype. In this review, we will discuss the properties of the EF B-cell pathway, its relationship to other effector B-cell populations, its role in autoimmune diseases, and its contribution to human SLE. Furthermore, we discuss the relationship of EF B cells with Age-Associated B cells (ABCs), a TLR-7-driven B-cell population that mediates murine autoimmune and antiviral responses., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Systemic Lupus Erythematosus (SLE) is characterized by B cells lacking IgD and CD27 (double negative; DN). We show that DN cell expansions reflected a subset of CXCR5 - CD11c + cells (DN2) representing pre-plasma cells (PC). DN2 cells predominated in African-American patients with active disease and nephritis, anti-Smith and anti-RNA autoantibodies. They expressed a T-bet transcriptional network; increased Toll-like receptor-7 (TLR7); lacked the negative TLR regulator TRAF5; and were hyper-responsive to TLR7. DN2 cells shared with activated naive cells (aNAV), phenotypic and functional features, and similar transcriptomes. Their PC differentiation and autoantibody production was driven by TLR7 in an interleukin-21 (IL-21)-mediated fashion. An in vivo developmental link between aNAV, DN2 cells, and PC was demonstrated by clonal sharing. This study defines a distinct differentiation fate of autoreactive naive B cells into PC precursors with hyper-responsiveness to innate stimuli, as well as establishes prominence of extra-follicular B cell activation in SLE, and identifies therapeutic targets., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Plasmodium falciparum malaria is a deadly infectious disease in which Abs play a critical role in naturally acquired immunity. However, the specificity and nature of Abs elicited in response to malaria are only partially understood. Autoreactivity and polyreactivity are common features of Ab responses in several infections and were suggested to contribute to effective pathogen-specific Ab responses. In this article, we report on the regulation of B cells expressing the inherently autoreactive VH4-34 H chain (identified by the 9G4 mAb) and 9G4 + plasma IgG in adults and children living in a P. falciparum malaria-endemic area in West Africa. The frequency of 9G4 + peripheral blood CD19 + B cells was similar in United States adults and African adults and children; however, more 9G4 + B cells appeared in classical and atypical memory B cell compartments in African children and adults compared with United States adults. The levels of 9G4 + IgG increased following acute febrile malaria but did not increase with age as humoral immunity is acquired or correlate with protection from acute disease. This was the case, even though a portion of 9G4 + B cells acquired phenotypes of atypical and classical memory B cells and 9G4 + IgG contained equivalent numbers of somatic hypermutations compared with all other VHs, a characteristic of secondary Ab repertoire diversification in response to Ag stimulation. Determining the origin and function of 9G4 + B cells and 9G4 + IgG in malaria may contribute to a better understanding of the varied roles of autoreactivity in infectious diseases., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Objective: The significance of distinct B cell abnormalities in primary Sjögren's syndrome (SS) remains to be established. We undertook this study to analyze the phenotype and messenger RNA (mRNA) transcript profiles of B cell subsets in patients with primary SS and to compare them with those in sicca syndrome patients and healthy controls., Methods: CD19+ B cells from 26 patients with primary SS, 27 sicca syndrome patients, and 22 healthy controls were analyzed by flow cytometry. Gene expression profiles of purified B cell subsets (from 3-5 subjects per group per test) were analyzed using Affymetrix gene arrays., Results: Patients with primary SS had lower frequencies of CD27+IgD- switched memory B cells and CD27+IgD+ unswitched memory B cells compared with healthy controls. Unswitched memory B cell frequencies were also lower in sicca syndrome patients and correlated inversely with serologic hyperactivity in both disease states. Further, unswitched memory B cells in primary SS had lower expression of CD1c and CD21. Gene expression analysis of CD27+ memory B cells separated patients with primary SS from healthy controls and identified a subgroup of sicca syndrome patients with a primary SS-like transcript profile. Moreover, unswitched memory B cell gene expression analysis identified 187 genes differentially expressed between patients with primary SS and healthy controls., Conclusion: A decrease in unswitched memory B cells with serologic hyperactivity is characteristic of both established primary SS and a subgroup of sicca syndrome, which suggests the value of these B cells both as biomarkers of future disease progression and for understanding disease pathogenesis. Overall, the mRNA transcript analysis of unswitched memory B cells suggests that their activation in primary SS takes place through innate immune pathways in the context of attenuated antigen-mediated adaptive signaling. Thus, our findings provide important insight into the mechanisms and potential consequences of decreased unswitched memory B cells in primary SS., (Copyright © 2014 by the American College of Rheumatology.)

Objective: To determine the prevalence of anti-apoptotic cell (anti-AC) antibodies with the 9G4 idiotype (9G4+) and the relationship between this and other known 9G4+ specificities and disease activity in patients with systemic lupus erythematosus (SLE)., Methods: Serum samples from 60 SLE patients and 40 healthy donors were incubated with apoptotic Jurkat cells and assayed by flow cytometry for the binding of 9G4+ antibodies. The samples were also tested for 9G4+ reactivity against naive B cells and total IgG and IgM anti-AC antibody reactivity., Results: The 9G4+ antibodies bound late ACs in sera from a majority of the SLE patients (60%) but in sera from only 2 healthy control subjects. Among samples with global IgM or IgG anti-AC antibodies, those with 9G4+ anti-AC antibodies predominated. Patients with high levels of 9G4+ anti-AC antibodies were more likely to have active disease. This was the case even in patients with IgG anti-AC antibodies or anti-double-stranded DNA antibodies. Patients with lupus nephritis were also more likely to have 9G4+ anti-AC antibodies. While 9G4+ reactivity to ACs often coincided with anti-B cell reactivity, some samples had distinct anti-AC or anti-B cell reactivity., Conclusion: The 9G4+ antibody represents a major species of anti-AC antibody in SLE serum, and this autoreactivity is associated with disease activity. The anti-AC reactivity of 9G4+ antibodies can be separated from the germline VH4-34-encoded anti-B cell autoreactivity. Our results indicate that ACs are an important antigenic source in SLE that positively selects B cells with intrinsic autoreactivity against other self antigens. This selection of 9G4+ B cells by ACs may represent an important step in disease progression., (Copyright © 2013 by the American College of Rheumatology.)

9G4(+) IgG Abs expand in systemic lupus erythematosus (SLE) in a disease-specific fashion and react with different lupus Ags including B cell Ags and apoptotic cells. Their shared use of VH4-34 represents a unique system to understand the molecular basis of lupus autoreactivity. In this study, a large panel of recombinant 9G4(+) mAbs from single naive and memory cells was generated and tested against B cells, apoptotic cells, and other Ags. Mutagenesis eliminated the framework-1 hydrophobic patch (HP) responsible for the 9G4 idiotype. The expression of the HP in unselected VH4-34 cells was assessed by deep sequencing. We found that 9G4 Abs recognize several Ags following two distinct structural patterns. B cell binding is dependent on the HP, whereas anti-nuclear Abs, apoptotic cells, and dsDNA binding are HP independent and correlate with positively charged H chain third CDR. The majority of mutated VH4-34 memory cells retain the HP, thereby suggesting selection by Ags that require this germline structure. Our findings show that the germline-encoded HP is compulsory for the anti-B cell reactivity largely associated with 9G4 Abs in SLE but is not required for reactivity against apoptotic cells, dsDNA, chromatin, anti-nuclear Abs, or cardiolipin. Given that the lupus memory compartment contains a majority of HP(+) VH4-34 cells but decreased B cell reactivity, additional HP-dependent Ags must participate in the selection of this compartment. This study represents the first analysis, to our knowledge, of VH-restricted autoreactive B cells specifically expanded in SLE and provides the foundation to understand the antigenic forces at play in this disease.

Regulation of B cell receptor signaling is essential for the development of specific immunity while retaining tolerance to self. Systemic lupus erythematosus (SLE) is characterized by a loss of B cell tolerance and the production of anti-self antibodies. Accompanying this break down in tolerance are alterations in B cell receptor signal transduction including elevated induced calcium responses and increased protein phosphorylation. Specific pathways that negatively regulate B cell signaling have been shown to be impaired in some SLE patients. These patients have reduced levels of the kinase Lyn in lipid raft microdomains and this reduction is inversely correlated with increased CD45 in lipid rafts. Function and expression of the inhibitory immunoglobulin receptor FcgammaRIIB is also reduced in Lupus IgM- CD27+ memory cells. Because the relative contribution of different memory and transitional B cell subsets can be abnormal in SLE patients, we believe studies targeted to well defined B cell subsets will be necessary to further our understanding of signaling abnormalities in SLE. Intracellular flow cytometric analysis of signaling is a useful approach to accomplish this goal.

Immunodominance refers to the restricted peptide specificity of T cells that are detectable after an adaptive immune response. For CD4 T cells, many of the mechanisms used to explain this selectivity suggest that events related to Ag processing play a major role in determining a peptide's ability to recruit CD4 T cells. Implicit in these models is the prediction that the molecular context in which an antigenic peptide is contained will impact significantly on its immunodominance. In this study, we present evidence that the selectivity of CD4 T cell responses to peptides contained within protein Ags is not detectably influenced by the location of the peptide in a given protein or the primary sequence of the protein that bears the test peptide. We have used molecular approaches to change the location of peptides within complex protein Ags and to change the flanking sequences that border the peptide epitope to now include a protease site, and find that immunodominance or crypticity of a peptide observed in its native protein context is preserved. Collectively, these results suggest immunodominance of peptides contained in complex Ags is due to an intrinsic factor of the peptide, based upon the affinity of that peptide for MHC class II molecules. These findings are discussed with regard to implications for vaccine design.

We have used TCR transgenic mice directed to different MHC class II-restricted determinants from the influenza virus hemagglutinin (HA) to analyze how specificity for self-peptides can shape CD4+CD25+ regulatory T (Treg) cell formation. We show that substantial increases in the number of CD4+CD25+ Treg cells can occur when an autoreactive TCR directed to a major I-E(d)-restricted determinant from HA develops in mice expressing HA as a self-Ag, and that the efficiency of this process is largely unaffected by the ability to coexpress additional TCR alpha-chains. This increased formation of CD4+CD25+ Treg cells in the presence of the self-peptide argues against models that postulate selective survival rather than induced formation as mechanisms of CD4+CD25+ Treg cell formation. In contrast, T cells bearing a TCR directed to a major I-A(d)-restricted determinant from HA underwent little or no selection to become CD4+CD25+ Treg cells in mice expressing HA as a self-Ag, correlating with inefficient processing and presentation of the peptide from the neo-self-HA polypeptide. These findings show that interactions with a self-peptide can induce thymocytes to differentiate along a pathway to become CD4+CD25+ Treg cells, and that peptide editing by DM molecules may help bias the CD4+CD25+ Treg cell repertoire away from self-peptides that associate weakly with MHC class II molecules.

The MHC-encoded cofactor DM catalyzes endosomal loading of peptides onto MHC class II molecules. Despite evidence from in vitro experiments that DM acts to selectively edit the repertoire of class II:peptide complexes, the consequence of DM expression in vivo, or a predictive pattern of DM activity in the specificity of CD4 T cell responses has remained unresolved. Therefore, to characterize DM function in vivo we used wild-type (WT) or DM-deficient (DM(-/-)) mice of the H-2(d) MHC haplotype and tested the hypothesis that DM promotes narrowing of the repertoire of class II:peptide complexes displayed by APC, leading to a correspondingly selective CD4 T cell response. Surprisingly, our results indicated that DM(-/-) mice do not exhibit a broadened CD4 T cell response relative to WT mice, but rather shift their immunodominance pattern to new peptides, a pattern associated with a change in class II isotype-restriction. Specifically, we found that CD4 T cell responses in WT mice were primarily restricted to the I-A class II molecule, whereas DM(-/-) mice recognize peptides in the context of I-E. The observed shift in isotype-restriction appeared to be due in part to a modification in the peripheral CD4 T cell repertoire available for peptide recognition.

Immunodominance refers to the restricted antigen specificity of T cells detected in the immune response after immunization with complex antigens. Despite the presence of many potential peptide epitopes within these immunogens, the elicited T-cell response apparently focuses on a very limited number of peptides. Over the last two decades, a number of distinct explanations have been put forth to explain this very restricted specificity of T cells, many of which suggest that endosomal antigen processing restricts the array of peptides available to recruit CD4 T cells. In this review, we present evidence from our laboratory that suggest that immunodominance in CD4 T-cell responses is primarily due to an intrinsic property of the peptide:class II complexes. The intrinsic kinetic stability of peptide:class II complexes controls DM editing within the antigen-presenting cells and thus the initial epitope density on priming dendritic cells. Additionally, we hypothesize that peptides that possess high kinetic stability interactions with class II molecules display persistence at the cell surface over time and will more efficiently promote T-cell signaling and differentiation than competing, lower-stability peptides contained within the antigen. We discuss this model in the context of the existing data in the field of immunodominance.

T cell priming to exogenous antigens reflects regulated antigen processing in dendritic cells, subsequent homing to lymph nodes, sustained interactions between T cells and antigen-bearing dendritic cells, and, ultimately, selective T cell activation and differentiation. In this study, we test the hypothesis that an intrinsic property of the class II:peptide complex is a key determinant that dictates the specificity of an emerging CD4 T cell response. We found that immunodominant peptides possess extremely long half-lives with class II molecules (t(1/2) > 150 hr), whereas cryptic peptides displayed half-lives of less than 10 hr. Furthermore, and most importantly, by using a peptide shuttle vector and four independent antigens, we demonstrate a direct, causative relationship between the half-life of peptide epitopes and their immunogenicity in vivo. Taken collectively, our results suggest the half-life of class II:peptide complexes is the primary parameter that dictates the ultimate hierarchy of the elicited T cell response.

Initial T cell activation in the context of different co-stimulatory receptors can influence subsequent lineage commitment into T(h) effector cell subtypes. Specifically, CD28 co-stimulation promotes T(h)2 differentiation, whereas leukocyte function-associated antigen-1 (LFA-1) co-stimulation promotes T(h)1 differentiation and inhibits T(h)2 differentiation. In this report, we have addressed the mechanism of LFA-1-mediated inhibition of T(h)2 responses. We show that co-stimulation through LFA-1 does not decrease early IL-4 secretion, but rather induces a loss in IL-4 responsiveness. T cells primed in the context of LFA-1 co-stimulation require a 5-fold increase in the concentration of IL-4 required to drive T(h)2 differentiation, which is not mediated by a loss in IL-4R expression. To determine whether LFA-1 co-stimulation impacts on proximal signaling from the IL-4R, we first identified a kinetic window where we could separate IL-4-driven T(h)2 differentiation from initial T cell priming. T cells were primed for 2 days under different co-stimulation conditions and re-cultured in the presence of IL-4. Subsequent T(h)2 differentiation was absolutely dependent on addition of IL-4. Proximal IL-4R signaling, as evidenced by tyrosine phosphorylation of signal transducer and activator of transcription-6 (STAT6), was not inhibited by initial co-stimulation through LFA-1, yet these T cells still required higher amounts of IL-4 and corresponding higher levels of STAT6 activation to up-regulate GATA-3 and induce T(h)2 differentiation. Thus, LFA-1 co-stimulation appears to interfere with GATA-3 expression downstream of STAT6. These results suggest that LFA-1 co-stimulation functions as a threshold modulator of T(h)2 differentiation, increasing the effective concentration of IL-4 required to drive T(h)2 responses.

Costimulation is one of several factors that influence the differentiation of CD4+ Th cell responses. Previously, we have shown that Ag presentation in the context of LFA-1 costimulation by fibroblasts transfected with class II and ICAM-1 (ProAd-ICAM) can drive naive CD4-positive T cells into cell cycle, but these T cells die by apoptosis 4-5 days after stimulation. In this report we show that the death of these cells can be prevented by the addition of exogenous IL-2 (20 U/ml) or by restimulation with Ag presented in the context of CD28 costimulation. Under these conditions, T cells go through extensive cell division and normal cell expansion. However, when T cells that have been primed by Ag presented in the context of LFA-1 costimulation are restimulated, they secrete IL-2 and IFN-gamma, but little or no IL-4. The inability of ProAd-ICAM-primed T cells to produce IL-4 was restored by the addition of IL-4 to the priming culture. However, IL-4 responses were not restored by representation of Ag in the context of CD28 costimulation, even as early as 24 h after priming with Ag presented by ProAd-ICAM cells. These findings suggest that differential expression of B7-1 and ICAM-1 by APCs during the initiation of immune responses may alter the differentiation of Th populations.