Highlights d PepSeq enables fully in vitro, highly multiplexed peptidebased antibody assays d Epitope mapping shows preexisting antibody reactivity to SARS-CoV-2 antigens d Antibodies cross-recognize endemic and pandemic antigens in the Spike S2 subunit d Cross-reactive antibodies raised by SARS-CoV-2 preferentially bind endemic homologs

Background Allergy, the most common disease of immune dysregulation, has a substantial genetic component that is poorly understood. While complete disruption of TCR signaling causes profound immunodeficiency, little is known about the consequences of inherited genetic variants that cause partial, quantitative decreases in particular TCR signaling pathways, despite their potential to dysregulate immune responses and cause immunopathology. Objective To elucidate how an inherited decrease in TCR signaling through CARD11, a critical scaffold protein that signals to NFκB transcription factors, results in spontaneous, selective accumulation of large numbers of Th2 cells. Methods ‘Unmodulated’ mice carry a Card11 single nucleotide variant (SNV) that decreases but does not abolish TCR/CD28 signaling to induce targets of NFκB. The consequences of this mutation on T cell subset formation in vivo were examined, and its effects within effector versus regulatory subsets were dissected by the adoptive transfer of wild-type cells, and by the examination of Foxp3-deficient unmodulated mice. Results Unlike the pathology-free boundary points of complete Card11 sufficiency or deficiency, unmodulated mice develop a specific allergic condition characterized by elevated IgE and dermatitis. The SNV partially decreases both the frequency of Foxp3+ T regulatory (Treg) cells and the efficiency of effector T cell formation in vivo. These intermediate effects combine to cause a gradual, selective expansion of Th2 cells. Conclusions Inherited reduction in the efficiency of TCR-NFκB signaling has graded effects on T cell activation and Foxp3+ Treg suppression that result in selective Th2 dysregulation and allergic disease.

Foxp3 ؉ regulatory T cells play a pivotal role in maintaining self-tolerance and immune homeostasis. In the absence of regulatory T cells, generalized immune activation and multiorgan T cell-driven pathology occurs. Although the phenomenon of immunologic control by Foxp3 ؉ regulatory T cells is well recognized, the comparative effect over different arms of the immune system has not been thoroughly investigated. Here, we generated a cohort of mice with a continuum of regulatory T-cell frequencies ranging from physiologic levels to complete deficiency. This titration of regulatory T-cell depletion was used to determine how different effector subsets are controlled. We found that in vivo Foxp3 ؉ regulatory T-cell frequency had a proportionate relationship with generalized T-cell activation and Th1 magnitude, but it had a surprising disproportionate relationship with Th2 magnitude. The asymmetric regulation was associated with efficient suppression of Th2 cells through additional regulations on the apoptosis rate in Th2 cells and not Th1 cells and could be replicated by CTLA4-Ig or anti-IL-2 Ab. These results indicate that the Th2 arm of the immune system is under tighter control by regulatory T cells than the Th1 arm, suggesting that Th2-driven diseases may be more responsive to regulatory T-cell manipulation. (Blood. 2011;118(7):1845-1853) IntroductionFoxp3 ϩ Regulatory T cells (Tregs) are a key modulator of immune system activation, with the ability to suppress the proliferation and cytokine production of CD4 ϩ and CD8 ϩ T cells. [1][2][3] Although Tregs require Ag-specific stimulation for activation, after activation the suppressive function acquired is Ag nonspecific. 4 This allows Tregs to act as a nonspecific rheostat on immune activation, decreasing the rate of spontaneous effector T-cell activation and thereby increasing dependence on pathogen-associated danger stimuli. 5 As such, Tregs not only prevent autoimmunity because of autoreactive T cells 6 but also reduce the activity of beneficial antipathogen 7 and antitumor 8 responses.The model of Tregs as indiscriminate suppressors is being challenged by data showing surprisingly sophisticated molecular underpinning of Treg suppression. Foxp3 ϩ Tregs use different molecular strategies to suppress T cells in different anatomical locations and to control different effector subpopulations. 9-14 On the receiving end of immune tolerance, there is a growing body of evidence that Th1 and Th2 cells have qualitative differences in sensitivity to intrinsic regulation. For example, Th1 cells have enhanced, and more rapid, activation-induced cell death (AICD). 15 In Th1 cells, AICD is mediated by Fas-FasL signaling and regulated by CD44, 16,17 whereas in Th2 cells AICD is mediated by granzyme B activity and regulated by VIP. 18,19 Likewise, Th1 cells are sensitive to endogenous galectin 1-induced cell death, whereas Th2 cells are resistant because of differential sialylation. 20 Because Treg cells have been reported to use both granzyme B and galectin 1 as regulatory med...

A high-resolution understanding of the antibody response to SARS-CoV-2 is important for the design of effective diagnostics, vaccines and therapeutics. However, SARS-CoV-2 antibody epitopes remain largely uncharacterized, and it is unknown whether and how the response may cross-react with related viruses. Here, we use a multiplexed peptide assay (‘PepSeq’) to generate an epitope-resolved view of reactivity across all human coronaviruses. PepSeq accurately detects SARS-CoV-2 exposure and resolves epitopes across the Spike and Nucleocapsid proteins. Two of these represent recurrent reactivities to conserved, functionally-important sites in the Spike S2 subunit, regions that we show are also targeted for the endemic coronaviruses in pre-pandemic controls. At one of these sites, we demonstrate that the SARS-CoV-2 response strongly and recurrently cross-reacts with the endemic virus hCoV-OC43. Our analyses reveal new diagnostic and therapeutic targets, including a site at which SARS-CoV-2 may recruit common pre-existing antibodies and with the potential for broadly-neutralizing responses.

CD1d-dependent NKT-cells represent a heterogeneous family of effector T-cells including CD4+CD8− and CD4−CD8− subsets, that respond to glycolipid antigens with rapid and potent cytokine production. NKT-cell development is regulated by a unique combination of factors, however very little is known about factors that control the development of NKT subsets. Here, we analyze a novel mouse strain (helpless) with a mis-sense mutation in the BTB-POZ domain of Zbtb7b and demonstrate that this mutation has dramatic, intrinsic effects on development of NKT-cell subsets. Although NKT-cell numbers are similar in Zbtb7b mutant mice, these cells are hyperproliferative and most lack CD4 and instead express CD8. Moreover, the majority of Zbtb7b mutant NKT-cells in the thymus are RORγt+ and a high frequency produce IL-17 while very few produce IFN-γ or other cytokines, sharply contrasting the profile of normal NKT-cells. Mice heterozygous for the helpless mutation also have reduced numbers of CD4+ NKT-cells and increased production of IL-17 without an increase in CD8+ cells, suggesting that Zbtb7b acts at multiple stages of NKT-cell development. These results reveal Zbtb7b as a critical factor genetically pre-determining the balance of effector subsets within the NKT-cell population.