A Virus-Specific Immune Rheostat in the Immunome of Patients Recovering From Mild COVID-19.

An accurate depiction of the convalescent COVID-19 immunome will help delineate the immunological milieu crucial for disease resolution and protection. Using mass cytometry, we characterized the immune architecture in patients recovering from mild COVID-19. We identified a virus-specific immune rheostat composed of an effector T (Teff) cell recall response that is balanced by the enrichment of a highly specialized regulatory T (Treg) cell subset. Both components were reactive against a peptide pool covering the receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein. We also observed expansion of IFNγ+ memory CD4+ T cells and virus-specific follicular helper T (TFH) cells. Overall, these findings pinpoint critical immune effector and regulatory mechanisms essential for a potent, yet harmless resolution of COVID-19 infection.

A subpopulation of high IL-21-producing CD4(+) T cells in Peyer's Patches is induced by the microbiota and regulates germinal centers.

The production of IL-21 by T follicular helper (Tfh) cells is vital in driving the germinal centre reaction and high affinity antibody formation. However, the degree of Tfh cell heterogeneity and function is not fully understood. We used a novel IL-21eGFP reporter mouse strain to analyze the diversity and role of Tfh cells. Through the analysis of GFP expression in lymphoid organs of IL-21eGFP mice, we identified a subpopulation of GFP(+), high IL-21 producing Tfh cells present only in Peyer's Patches. GFP(+)Tfh cells were found to be polyclonal and related to GFP(-)Tfh cells of Peyer's Patches in TCR repertoire composition and overall gene expression. Studies on the mechanisms of induction of GFP(+)Tfh cells demonstrated that they required the intestinal microbiota and a diverse repertoire of CD4(+) T cells and B cells. Importantly, ablation of GFP(+) cells resulted in a reduced frequency of Peyer's Patches IgG1 and germinal center B cells in addition to small but significant shifts in gut microbiome composition. Our work highlights the diversity among IL-21 producing CD4(+) Tfh cells, and the interrelationship between the intestinal bacteria and Tfh cell responses in the gut.

IRF8 directs stress-induced autophagy in macrophages and promotes clearance of Listeria monocytogenes.

Autophagy, activated by many stresses, plays a critical role in innate immune responses. Here we show that interferon regulatory factor 8 (IRF8) is required for the expression of autophagy-related genes in dendritic cells. Furthermore in macrophages, IRF8 is induced by multiple autophagy-inducing stresses, including IFNγ and Toll-like receptor stimulation, bacterial infection, starvation and by macrophage colony-stimulating factor. IRF8 directly activates many genes involved in various steps of autophagy, promoting autophagosome formation and lysosomal fusion. Consequently, Irf8(-/-) macrophages are deficient in autophagic activity, and excessively accumulate SQSTM1 and ubiquitin-bound proteins. We show that clearance of Listeria monocytogenes in macrophages requires IRF8-dependent activation of autophagy genes and subsequent autophagic capturing and degradation of Listeria antigens. These processes are defective in Irf8(-/-) macrophages where uninhibited bacterial growth ensues. Together these data suggest that IRF8 is a major autophagy regulator in macrophages, essential for macrophage maturation, survival and innate immune responses.

The distinctive germinal center phase of IgE+ B lymphocytes limits their contribution to the classical memory response.

The mechanisms involved in the maintenance of memory IgE responses are poorly understood, and the role played by germinal center (GC) IgE(+) cells in memory responses is particularly unclear. IgE(+) B cell differentiation is characterized by a transient GC phase, a bias toward the plasma cell (PC) fate, and dependence on sequential switching for the production of high-affinity IgE. We show here that IgE(+) GC B cells are unfit to undergo the conventional GC differentiation program due to impaired B cell receptor function and increased apoptosis. IgE(+) GC cells fail to populate the GC light zone and are unable to contribute to the memory and long-lived PC compartments. Furthermore, we demonstrate that direct and sequential switching are linked to distinct B cell differentiation fates: direct switching generates IgE(+) GC cells, whereas sequential switching gives rise to IgE(+) PCs. We propose a comprehensive model for the generation and memory of IgE responses.

Cross-roads in the lung: immune cells and tissue interactions as determinants of allergic asthma.

Allergic asthma is a chronic disease of the lung characterized by underlying Th2- and IgE-mediated inflammation, structural alterations of the bronchial wall, and airway hyperresponsiveness. Initial allergic sensitization and later development of chronic disease are determined by close interactions between lung structural cells and the resident and migratory immune cells in the lung. Epithelial cells play a crucial role in allergic sensitization by directly influencing dendritic cells induction of tolerant or effector T cells and production of type 2 cytokines by innate immune cells. During chronic disease, the bronchial epithelium, stroma, and smooth muscle become structurally and functionally altered, contributing to the perpetuation of tissue remodeling. Thus, targeting tissue-driven pathology in addition to inflammation may increase the effectiveness of asthma treatment.

Codon optimization of the tat antigen of human immunodeficiency virus type 1 generates strong immune responses in mice following genetic immunization.

DNA vaccines have been successful in eliciting potent immune responses in mice. Their efficiency, however, is restricted in larger animals. One reason for the limited performance of the DNA vaccines is the lack of molecular strategies to enhance immune responses. Additionally, genes directly cloned from pathogenic organisms may not be efficiently translated in a heterologous host expression system as a consequence of codon bias. To evaluate the influence of codon optimization on the immune response, we elected to use the Tat antigens of human immunodeficiency virus type 1 (HIV-1) (subtype C) and HIV-2, as these viral antigens are poorly immunogenic in natural infection and in experimental immunization and they are functionally important in viral infectivity and pathogenesis. Substituting codons that are optimally used in the mammalian system, we synthetically assembled Tat genes and compared them with the wild-type counterparts in two different mouse strains. Codon-optimized Tat genes induced qualitatively and quantitatively superior immune responses as measured in a T-cell proliferation assay, enzyme-linked immunospot assay, and chromium release assay. Importantly, while the wild-type genes promoted a mixed Th1-Th2-type cytokine profile, the codon-optimized genes induced a predominantly Th1 profile. Using a pepscan strategy, we mapped an immunodominant T-helper epitope to the core and basic domains of HIV-1 Tat. We also identified cross-clade immune responses between HIV-1 subtype B and C Tat proteins mapped to this T-helper epitope. Developing molecular strategies to optimize the immunogenicity of DNA vaccines is critical for inducing strong immune responses, especially to antigens like Tat. Our identification of a highly conserved T-helper epitope in the first exon of HIV-1 Tat of subtype C and the demonstration of a cross-clade immune response between subtypes B and C are important for a more rational design of an HIV vaccine.

Codon optimization and ubiquitin conjugation of human immunodeficiency virus-1 Tat lead to enhanced cell-mediated immune responses.

The transactivator protein, Tat, is a potential candidate for developing a vaccine against human immunodeficiency virus (HIV-1). Since Tat is not immunodominant, especially when delivered as a genetic vaccine, we expressed codon-optimized subtype-C Tat as a molecular conjugate of ubiquitin, to elicit antigen-specific cell-mediated immune responses. Immunization of mice with different ubiquitin-Tat constructs elicited a strong cellular, but not a humoral, immune response. The combination of codon-optimization and ubiquitin-mediated processing of Tat induced a Th-1 type cellular immune response that was detectable without in vitro stimulation, suggesting its potential utility for destruction of virus-infected cells via CTL-mediated lysis. Preliminary attempts at characterizing the immunodominant regions identified a novel T-helper epitope within the core domain of Tat.

Tat protein of human immunodeficiency virus type 1 subtype C strains is a defective chemokine.

Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) is correlated with increased monocyte migration to the brain, and the incidence of HAD among otherwise asymptomatic subjects appears to be lower in India than in the United States and Europe (1 to 2% versus 15 to 30%). Because of the genetic differences between HIV-1 strains circulating in these regions, we sought to identify viral determinants associated with this difference. We targeted Tat protein for these studies in view of its association with monocyte chemotactic function. Analyses of Tat sequences representing nine subtypes revealed that at least six amino acid residues are differentially conserved in subtype C Tat (C-Tat). Of these, cysteine (at position 31) was highly (>99%) conserved in non-subtype C viruses and more than 90% of subtype C viruses encoded a serine. We hypothesized a compromised chemotactic function of C-Tat due to the disruption of CC motif and tested it with the wild type C-Tat (CS) and its two isogenic variants (CC and SC) derived by site-directed mutagenesis. We found that the CS natural variant was defective for monocyte chemotactic activity without a loss in the transactivation property. While the CC mutant is functionally competent for both the functions, in contrast, the SC mutant was defective in both. Therefore, the loss of the C-Tat chemotactic property may underlie the reduced incidence of HAD; although not presenting conclusive evidence, this study provides the first evidence for a potential epidemiologic phenomenon associated with biological differences in the subtype C viruses.