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Background: While vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) provides significant protection from coronavirus disease 2019, the protection afforded to individuals with chronic lung disease is less well established. This study seeks to understand how chronic lung disease impacts SARS-CoV-2 vaccine-elicited immunity., Methods: Deep immune phenotyping of humoral and cell-mediated responses to the SARS-CoV-2 vaccine was performed in patients with asthma, COPD and interstitial lung disease (ILD) compared to healthy controls., Results: 48% of vaccinated patients with chronic lung diseases had reduced antibody titres to the SARS-CoV-2 vaccine antigen relative to healthy controls. Vaccine antibody titres were significantly reduced among asthma (p<0.035), COPD (p<0.022) and a subset of ILD patients as early as 3-4 months after vaccination, correlating with decreased vaccine-specific memory B-cells in circulation. Vaccine-specific memory T-cells were significantly reduced in patients with asthma (CD8 + p<0.004; CD4 + p<0.023) and COPD (CD8 + p<0.008) compared to healthy controls. Impaired T-cell responsiveness was also observed in a subset of ILD patients (CD8 + 21.4%; CD4 + 42.9%). Additional heterogeneity between healthy and disease cohorts was observed among bulk and vaccine-specific follicular T-helper cells., Conclusions: Deep immune phenotyping of the SARS-CoV-2 vaccine response revealed the complex nature of vaccine-elicited immunity and highlights the need for more personalised vaccination schemes in patients with underlying lung conditions., Competing Interests: Conflict of interest: Authors report no competing financial interests., (Copyright ©The authors 2023.)

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.

Resolution of inflammation is an active process that is tightly regulated to achieve repair and tissue homeostasis. In the absence of resolution, persistent inflammation underlies the pathogenesis of chronic lung disease such as chronic obstructive pulmonary disease (COPD) with recurrent exacerbations. Over the course of inflammation, macrophage programming transitions from pro-inflammatory to pro-resolving, which is in part regulated by the nuclear receptor Peroxisome Proliferator-Activated Receptor γ (PPARγ). Our previous work demonstrated an association between Fatty Acid Binding Protein 5 (FABP5) expression and PPARγ activity in peripheral blood mononuclear cells of healthy and COPD patients. However, a role for FABP5 in macrophage programming has not been examined. Here, using a combination of in vitro and in vivo approaches, we demonstrate that FABP5 is necessary for PPARγ activation. In turn, PPARγ acts directly to increase FABP5 expression in primary human alveolar macrophages. We further illustrate that lack of FABP5 expression promotes a pro-inflammatory macrophage programming with increased secretion of pro-inflammatory cytokines and increased chromatin accessibility for pro-inflammatory transcription factors (e.g., NF-κB and MAPK). And finally, real-time cell metabolic analysis using the Seahorse technology shows an inhibition of oxidative phosphorylation in FABP5-deficient macrophages. Taken together, our data indicate that FABP5 and PPARγ reciprocally regulate each other's expression and function, consistent with a novel positive feedback loop between the two factors that mediates macrophage pro-resolving programming. Our studies highlight the importance of defining targets and regulatory mechanisms that control the resolution of inflammation and may serve to inform novel interventional strategies directed towards COPD., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

The newly emerging variants of SARS-CoV-2 from India (Delta variant) and South America (Lambda variant) have led to a higher infection rate of either vaccinated or unvaccinated people. We found that sera from Pfizer-BioNTech vaccine remain high reactivity toward the receptor binding domain (RBD) of Delta variant while it drops dramatically toward that of Lambda variant. Interestingly, the overall titer of antibodies of Pfizer-BioNTech vaccinated individuals drops 3-fold after 6 months, which could be one of major reasons for breakthrough infections, emphasizing the importance of potential third boost shot. While a therapeutic antibody, Bamlanivimab, decreases binding affinity to Delta variant by ~20 fold, it fully lost binding to Lambda variant. Structural modeling of complexes of RBD with human receptor, Angiotensin Converting Enzyme 2 (ACE2), and Bamlanivimab suggest the potential basis of the change of binding. The data suggest possible danger and a potential surge of Lambda variant in near future.

We generated several versions of the receptor binding domain (RBD) of the Spike protein with mutations existing within newly emerging variants from South Africa and Brazil. We found that the mutant RBD with K417N, E484K, and N501Y exchanges has higher binding affinity to the human receptor compared to the wildtype RBD. This mutated version of RBD also completely abolishes the binding to a therapeutic antibody, Bamlanivimab, in vitro .

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.

A paradox in monoclonal antibody (mAb) therapy is that despite the well-documented tolerogenic properties of deaggregated IgG, most therapeutic IgG mAb induce anti-mAb responses. To analyze CD4 T cell reactions against IgG in various physical states, we developed an adoptive transfer model using CD4+ T cells specific for a Vκ region-derived peptide in the hapten-specific IgG mAb 36-71. We found that heat-aggregated or immune complexes (IC) of mAb 36-71 elicited anti-idiotypic (anti-Id) antibodies, while the deaggregated form was tolerogenic. All 3 forms of mAb 36-71 induced proliferation of cognate CD4+ T cells, but the aggregated and immune complex forms drove more division cycles and induced T follicular helper cells (TFH) development more effectively than did the deaggregated form. These responses occurred despite no adjuvant and no or only trace levels of endotoxin in the preparations. Physical analyses revealed large differences in micron- and nanometer-sized particles between the aggregated and IC forms. These differences may be functionally relevant, as CD4+ T cell proliferation to aggregated, but not IC mAb 36-71, was nearly ablated upon peritoneal injection of B cell-depleting antibody. Our results imply that, in addition to denatured aggregates, immune complexes formed in vivo between therapeutic mAb and their intended targets can be immunogenic., Competing Interests: The authors have declared that no competing interests exist.

We previously reported that selective ablation of certain γδ T cell subsets, rather than removal of all γδ T cells, strongly affects serum Ab levels in nonimmunized mice. This type of manipulation also changed T cells, including residual γδ T cells, revealing some interdependence of γδ T cell populations. For example, in mice lacking Vγ4(+) and Vγ6(+) γδ T cells (B6.TCR-Vγ4(-/-)/6(-/-)), we observed expanded Vγ1(+) cells, which changed in composition and activation and produced more IL-4 upon stimulation in vitro, increased IL-4 production by αβ T cells as well as spontaneous germinal center formation in the spleen, and elevated serum Ig and autoantibodies. We therefore examined B cell populations in this and other γδ-deficient mouse strains. Whereas immature bone marrow B cells remained largely unchanged, peripheral B cells underwent several changes. Specifically, transitional and mature B cells in the spleen of B6.TCR-Vγ4(-/-)/6(-/-) mice and other peripheral B cell populations were diminished, most of all splenic marginal zone (MZ) B cells. However, relative frequencies and absolute numbers of Ab-producing cells, as well as serum levels of Abs, IL-4, and BAFF, were increased. Cell transfers confirmed that these changes are directly dependent on the altered γδ T cells in this strain and on their enhanced potential of producing IL-4. Further evidence suggests the possibility of direct interactions between γδ T cells and B cells in the splenic MZ. Taken together, these data demonstrate the capability of γδ T cells of modulating size and productivity of preimmune peripheral B cell populations., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Somatic gene rearrangement generates a diverse repertoire of B cells, many which have receptors possessing a range of affinities for self-Ag. Newly generated B cells express high and relatively uniform amounts of surface IgM (sIgM), while follicular (FO) B cells express sIgM at widely varying levels. It is plausible, therefore, that downmodulation of sIgM serves as a mechanism to maintain weakly self-reactive B cells in a responsive state by decreasing their avidity for self-Ag. We tested this hypothesis by performing comparative functional tests with FO IgM(hi) and IgM(lo) B cells from the unrestricted repertoire of WT C57BL/6 mice. We found that FO IgM(lo) B cells mobilized Ca(2+) equivalently to IgM(hi) B cells when the same number of sIgM molecules was engaged. In agreement, FO IgM(lo) B cells were functionally competent to produce an antibody response following adoptive transfer. The FO IgM(lo) cell population had elevated levels of Nur77 transcript, and was enriched with nuclear-reactive specificities. Hybridoma sampling revealed that these B-cell receptors were of low affinity. Collectively, these results suggest that sIgM downmodulation by low-affinity, self-reactive B cells preserves their immunocompetence and circumvents classical peripheral tolerance mechanisms that would otherwise reduce diversity within the B cell compartment., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Our laboratory investigates systemic autoimmune disease in the context of mouse models of systemic lupus erythematosus (SLE). SLE is associated with high titers of serum autoantibodies of the IgG class that are predominantly directed against nuclear antigens, with pathological manifestations that are considered by many to be characteristic of an immune-complex mediated disease. In this review, we focus on the known and potential roles of somatic mutagenesis in SLE. We will argue that anti-nuclear antibodies (ANA) arise predominantly from nonautoreactive B cells that are transformed into autoreactive cells by the process of somatic hypermutation (SHM), which is normally associated with affinity maturation during the germinal center reaction. We will also discuss the role of SHM in creating antigenic peptides in the V region of the B cell receptor (BCR) and its potential to open an avenue of unregulated T cell help to autoreactive B cells. Finally, we will end this review with new experimental evidence suggesting that spontaneous somatic mutagenesis of genes that regulate B cell survival and activation is a rate-limiting causative factor in the development of ANA.

Autoreactive anergic B lymphocytes are considered to be dangerous because of their potential for activation and recruitment into autoimmune responses. However, they persist for days and constitute ∼5% of the B cell pool. We assessed their functional potential in the Ars/A1 transgene model, where anergic B cells express a dual-reactive Ag receptor that binds, in addition to a self-Ag, the hapten p-azophenylarsonate (Ars). When Ars/A1 B cells were transferred into adoptive recipients that were immunized with foreign proteins covalently conjugated with Ars, endogenous IgG immune responses to both were selectively and severely diminished, and the development of T helper cells was impaired. Approximately 95% inhibition of the anti-Ars response was attained with ∼4000 transferred Ars/A1 B cells through redundant mechanisms, one of which depended on their expression of MHC class II but not upon secretion of IL-10 or IgM. This Ag-specific suppressive activity implicates the autoreactive anergic B cell as an enforcer of immunological tolerance to self-Ags.

Linked recognition of Ag by B and T lymphocytes is ensured in part by a state of tolerance acquired by CD4 T cells to germline-encoded sequences within the B cell Ag receptor (BCR). We sought to determine how such tolerance is attained when a peptide from the BCR variable (V) region is expressed by small numbers of B cells as it is in the physiological state. Mixed bone marrow (BM) chimeras were generated using donor BM from mice with B cells that expressed a transgene (Tg)-encoded κ L chain and BM from TCR Tg mice in which the CD4 T cells (CA30) were specific for a Vκ peptide encoded by the κTg. In chimeras where few B cells express the κTg, many CA30 cells were deleted in the thymus. However, a substantial fraction survived to the CD4 single-positive stage. Among single-positive CA30 thymocytes, few reached maturity and migrated to the periphery. Maturation was strongly associated with, and likely promoted by, expression of an endogenous TCR α-chain. CD4(+) CA30 cells that reached peripheral lymphoid tissues were Ag-experienced and anergic, and some developed into regulatory cells. These findings reveal several checkpoints and mechanisms that enforce a state of self-tolerance in developing T cells specific for BCR V region sequences, thus ensuring that T cell help to B cells occurs through linked recognition of foreign Ag.

Systemic lupus erythematosus (SLE) is characterized by high-avidity IgG antinuclear antibodies (ANAs) that are almost certainly products of T cell-dependent immune responses. Whether critical amino acids in the third complementarity-determining region (CDR3) of the ANA originate from V(D)J recombination or somatic hypermutation (SHM) is not known. We studied a mouse model of SLE in which all somatic mutations within ANA V regions, including those in CDR3, could be unequivocally identified. Mutation reversion analyses revealed that ANA arose predominantly from nonautoreactive B cells that diversified immunoglobulin genes via SHM. The resolution afforded by this model allowed us to demonstrate that one ANA clone was generated by SHM after a V(H) gene replacement event. Mutations producing arginine substitutions were frequent and arose largely (66%) from base changes in just two codons: AGC and AGT. These codons are abundant in the repertoires of mouse and human V genes. Our findings reveal the predominant role of SHM in the development of ANA and underscore the importance of self-tolerance checkpoints at the postmutational stage of B cell differentiation.

T cell-dependent immune responses generate long-lived plasma cells and memory B cells, both of which express hypermutated Ab genes. The relationship between these cell types is not entirely understood. Both appear to emanate from the germinal center reaction, but it is unclear whether memory cells evolve while obligatorily generating plasma cells by siblings under all circumstances. In the experiments we report, plasma cell development was functionally segregated from memory cell development by a series of closely spaced injections of Ag delivered during the period of germinal center development. The injection series elevated serum Ab of low affinity, supporting the idea that a strong Ag signal drives plasma cell development. At the same time, the injection series produced a distinct population of affinity/specificity matured memory B cells that were functionally silent, as manifested by an absence of corresponding serum Ab. These cells could be driven by a final booster injection to develop into Ab-forming cells. This recall response required that a rest period precede the final booster injection, but a pause of only 4 days was sufficient. Our results support a model of memory B cell development in which extensive affinity/specificity maturation can take place within a B cell clone under some circumstances in which a concomitant generation of Ab-forming cells by siblings does not take place.

Activation of the innate immune system promotes polyclonal antibody secretion to eliminate invading pathogens. Inherent in this process is the potential to activate autoreactive B cells and induce autoimmunity. We showed previously that TLR-stimulated dendritic cells and macrophages regulate B cell tolerance to Smith antigen, in part through the secretion of interleukin-6 (IL-6). In this manuscript, we show that neutralization of IL-6 fails to abrogate macrophage-mediated repression and identify soluble CD40 ligand (CD40L) as a second repressive factor secreted by macrophages. CD40L selectively repressed Ig secretion by chronically antigen-experienced (anergic) immunoglobulin transgenic and nontransgenic B cells but not by transiently stimulated B cells. The importance of macrophages in maintaining B cell tolerance was apparent in lupus-prone MRL/lpr mice. Compared with C57BL/6 mice, macrophages from MRL/lpr mice were significantly less efficient at repressing immunoglobulin secretion coincident with diminished IL-6 and CD40 ligand production. These data indicate that macrophages regulate autoreactive B cells by secreting repressive factors that prohibit terminal differentiation of B cells. The regulation of autoreactive B cells by macrophages is diminished in lupus-prone mice suggesting a role in autoimmunity.

The contribution of anergy to silencing of autoreactive B cells in physiologic settings is unknown. By comparing anergic and nonanergic immunoglobulin-transgenic mouse strains, we defined a set of surface markers that were used for presumptive identification of an anergic B cell cohort within a normal repertoire. Like anergic transgenic B cells, these physiologic anergic cells exhibited high basal intracellular free calcium and did not mobilize calcium, initiate tyrosine phosphorylation, proliferate, upregulate activation markers, or mount an immune response upon antigen-receptor stimulation. Autoreactive B cells were overrepresented in this cohort. On the basis of the frequency and lifespan of these cells, it appears that as many as 50% of newly produced B cells are destined to become anergic. In conclusion, our findings indicate that anergy is probably the primary mechanism by which autoreactive B cells are silenced. Thus maintenance of the unresponsiveness of anergic cells is critical for prevention of autoimmunity.

The BCR V region has been implicated as a potential avenue of T cell help for autoreactive B cells in systemic lupus erythematosus. In principle, either germline-encoded or somatically generated sequences could function as targets of such help. Preceding studies have indicated that class II MHC-restricted T cells in normal mice attain a state tolerance to germline-encoded Ab diversity. In this study, we tested whether this tolerance is intact in systemic lupus erythematosus-prone (New Zealand Black x SWR)F1 mice (SNF1). Using a hybridoma sampling approach, we found that SNF1 T cells were tolerant to germline-encoded Ab sequences. Specifically, they were tolerant to germline-encoded sequences derived from a lupus anti-chromatin Ab that arose spontaneously in this strain. This was true both for diseased and prediseased mice. Thus, there does not appear to be a global defect in T cell tolerance to Ab V regions in this autoimmune-prone strain either before or during autoimmune disease.

Antibody diversity creates an immunoregulatory challenge for T cells that must cooperate with B cells, yet discriminate between self and nonself. To examine the consequences of T cell reactions to the B cell receptor (BCR), we generated a transgenic (Tg) line of mice expressing a T cell receptor (TCR) specific for a kappa variable region peptide in monoclonal antibody (mAb) 36-71. The kappa epitope was originally generated by a pair of somatic mutations that arose naturally during an immune response. By crossing this TCR Tg mouse with mice expressing the kappa chain of mAb 36-71, we found that kappa-specific T cells were centrally deleted in thymi of progeny that inherited the kappaTg. Maternally derived kappaTg antibody also induced central deletion. In marked contrast, adoptive transfer of TCR Tg T cells into kappaTg recipients resulted in T and B cell activation, lymphadenopathy, splenomegaly, and the production of IgG antichromatin antibodies by day 14. In most recipients, autoantibody levels increased with time, Tg T cells persisted for months, and a state of lupus nephritis developed. Despite this, Tg T cells appeared to be tolerant as assessed by severely diminished proliferative responses to the Vkappa peptide. These results reveal the importance of attaining central and peripheral T cell tolerance to BCR V regions. They suggest that nondeletional forms of T tolerance in BCR-reactive T cells may be insufficient to preclude helper activity for chromatin-reactive B cells.

Memory B cells of mice with Ig mu transgenes often carry transgene copies that have moved into the Igh locus via somatic translocation. This phenomenon has been attributed to a selection pressure for somatic hypermutations, which generally are observed at much higher frequencies in translocated copies than in ectopic copies. We tested this idea by immunizing Ig-mu transgenic mice in a manner designed to select B cells that required only one V(H) mutation for a switch in antigenic specificity and recruitment into the memory pool. Despite the minimal mutation requirement, hybridomas carrying somatic translocations to the Igh locus were obtained. Importantly, this occurred despite the fact that translocated and untranslocated mu-transgenes were mutated comparably. Evidently, a strong selection advantage was conferred upon B cells by the somatic translocations. Among the hybridomas, translocated mu-transgenes were active, while ectopic mu-transgenes were uniformly silent. The translocated copy that had conferred an affinity-based selection advantage was expressed at the highest level. Moreover, translocated copies were differentially expressed among hybridoma members, which belonged to a common post-mutational lineage. This suggests that adjustments in transgene expression levels had occurred during memory cell development. These results indicate that, apart from their potential influences on somatic hypermutagenesis and class switch recombination, elements in the Igh locus promote the selection of memory B cells in another way, possibly by regulating the level of Ig expression at various stages of antigen-driven differentiation.

Ig variable (V) region genes are subjected to a somatic hypermutation process as B lymphocytes participate in immune reactions to protein Ags. Although little is known regarding the mechanism of mutagenesis, a consistent hierarchy of trinucleotide target preferences is evident. Analysis of trinucleotide regional distributions predicted and we now empirically confirm the surprising finding that the framework 2 region of kappa V region genes is highly mutable despite its importance to the structural integrity and function of the Ab molecule. Interestingly, much of this mutability appears to be focused on the third codon position where synonymous substitutions are most likely to occur. We also observed a trend for high predicted mutability for codon positions 1 and 2 in complementarity-determining regions. Consequently, amino acid replacements should occur at a higher rate in complementarity-determining regions than in framework regions due to the distribution and subsequent targeting of microsequences by the mutation mechanism. Our results reveal a subtle tier of V region gene evolution in which DNA sequence has been molded to direct mutations to specific base positions within codons in a manner that minimizes damage and maximizes the benefits of the somatic hypermutation process.

Available evidence indicates that B cell tolerance is attained by receptor editing, anergy, or clonal deletion. Here, we describe a p-azophenylarsonate (Ars)-specific immunoglobulin transgenic mouse in which B cells become anergic as a consequence of cross-reaction with autoantigen in the bone marrow. Developing bone marrow B cells show no evidence of receptor editing but transiently upregulate activation markers and appear to undergo accelerated development. Mature B cells are present in normal numbers but are refractory to BCR-mediated induction of calcium mobilization, tyrosine phosphorylation, and antibody responses. Activation marker expression and acquisition of the anergic phenotype is prevented in bone marrow cultures by monovalent hapten. In this model, it appears that induction of anergy in B cells can be prevented by monovalent hapten competing with autoantigen for the binding site.

Somatic mutations are not distributed randomly throughout Ab V region genes. A sequence-specific target bias is revealed by a defined hierarchy of mutability among di- and trinucleotide sequences located within Ig intronic DNA. Here we report that the di- and trinucleotide mutability preference pattern is shared by mouse intronic JH and Jkappa clusters and by human VH genes, suggesting that a common mutation mechanism exists for all Ig V genes of both species. Using di- and trinucleotide target preferences, we performed a comprehensive analysis of human and murine germline V genes to predict regional mutabilities. Heavy chain genes of both species exhibit indistinguishable patterns in which complementarity-determining region 1 (CDR1), CDR2, and framework region 3 (FR3) are predicted to be more mutable than FR1 and FR2. This prediction is borne out by empirical mutation data from nonproductively rearranged human VH genes. Analysis of light chain genes in both species also revealed a common, but unexpected, pattern in which FR2 is predicted to be highly mutable. While our analyses of nonfunctional Ig genes accurately predicts regional mutation preferences in VH genes, observed relative mutability differences between regions are more extreme than expected. This cannot be readily accounted for by nascent mRNA secondary structure or by a supplemental gene conversion mechanism that might favor nucleotide replacements in CDR. Collectively, our data support the concept of a common mutation mechanism for heavy and light chain genes of mice and humans with regional bias that is qualitatively, but not quantitatively, accounted for by short nucleotide sequence composition.