Chronic inflammation, neutrophil activity, and autoreactivity splits long COVID.

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.

Dysregulated naive B cells and de novo autoreactivity in severe COVID-19.

Severe SARS-CoV-2 infection1 has been associated with highly inflammatory immune activation since the earliest days of the COVID-19 pandemic2-5. More recently, these responses have been associated with the emergence of self-reactive antibodies with pathologic potential6-10, although their origins and resolution have remained unclear11. Previously, we and others have identified extrafollicular B cell activation, a pathway associated with the formation of new autoreactive antibodies in chronic autoimmunity12,13, 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.

Relaxed peripheral tolerance drives broad de novo autoreactivity in severe COVID-19.

An emerging feature of COVID-19 is the identification of autoreactivity in patients with severe disease that may contribute to disease pathology, however the origin and resolution of these responses remain unclear. Previously, we identified strong extrafollicular B cell activation as a shared immune response feature between both severe COVID-19 and patients with advanced rheumatic disease. In autoimmune settings, this pathway is associated with relaxed peripheral tolerance in the antibody secreting cell compartment and the generation of de novo autoreactive responses. Investigating these responses in COVID-19, we performed single-cell repertoire analysis on 7 patients with severe disease. In these patients, we identify the expansion of a low-mutation IgG1 fraction of the antibody secreting cell compartment that are not memory derived, display low levels of selective pressure, and are enriched for autoreactivity-prone IGHV4-34 expression. Within this compartment, we identify B cell lineages that display specificity to both SARS-CoV-2 and autoantigens, including pathogenic autoantibodies against glomerular basement membrane, and describe progressive, broad, clinically relevant autoreactivity within these patients correlated with disease severity. Importantly, we identify anti-carbamylated protein responses as a common hallmark and candidate biomarker of broken peripheral tolerance in severe COVID-19. Finally, we identify the contraction of this pathway upon recovery, and re-establishment of tolerance standards coupled with a concomitant loss of acute-derived ASCs irrespective of antigen specificity. In total, this study reveals the origins, breadth, and resolution of acute-phase autoreactivity in severe COVID-19, with significant implications in both early interventions and potential treatment of patients with post-COVID sequelae.

Diversity of Salmonella Typhi-responsive CD4 and CD8 T cells before and after Ty21a typhoid vaccination in children and adults.

Typhoid fever is a life-threatening disease caused by the human-restricted pathogen Salmonella enterica serovar Typhi (S. Typhi). The oral live attenuated Ty21a typhoid vaccine protects against this severe disease by eliciting robust, multifunctional cell-mediated immunity (CMI), shown to be associated with protection in wild-type S. Typhi challenge studies. Ty21a induces S. Typhi-responsive CD8+ and CD4+ T cells but little is known about the response to this vaccine in children. To address this important gap in knowledge, we have used mass cytometry to analyze pediatric and adult pre- and post-Ty21a vaccination CMI in an autologous S. Typhi antigen presentation model. Here, using conventional supervised analytical tools, we show adult T cells are more multifunctional at baseline than those obtained from children. Moreover, pediatric and adult T cells respond similarly to Ty21a vaccination, but adult responders remain more multifunctional. The use of the unsupervised dimensionality reduction tool tSNE (t-distributed Stochastic Neighbor Embedding) allowed us to confirm these findings, as well as to identify increases and decreases in well-defined specific CD4+ and CD8+ T-cell populations that were not possible to uncover using the conventional gating strategies. These findings evidenced age-associated maturation of multifunctional S. Typhi-responsive T-cell populations, including those which we have previously shown to be associated with protection from, and/or delayed onset of, typhoid disease. These findings are likely to play an important role in improving pediatric vaccination strategies against S. Typhi and other enteric pathogens.

Characteristics of regulatory T-cell populations before and after Ty21a typhoid vaccination in children and adults.

Typhoid fever, caused by the pathogen Salmonella enterica serovar Typhi (S. Typhi), is a serious global health concern. Challenge studies with wild type S. Typhi identified associations between gut-homing regulatory T cells (Treg) and development of typhoid disease. Whether oral live-attenuated Ty21a vaccination induces gut-homing Treg remains unclear. Here, we analyze pediatric and adult Treg pre- and post-Ty21a vaccination in an autologous S. Typhi-antigen presentation model to address this knowledge gap. We show that peripheral memory Treg populations change from childhood to adulthood, but not following Ty21a vaccination. Unsupervised dimensionality reduction with t-distributed stochastic neighbor embedding (tSNE) identifies homing, memory, and functional features which evidence age-associated maturation of multifunctional S. Typhi-responsive Treg, which were not impacted by Ty21a vaccination. These findings improve understanding of pediatric regulatory T cells, while identifying age-related differences in S. Typhi-responsive Treg, which may aid in the development of improved pediatric vaccination strategies against S. Typhi.

Age-Associated Heterogeneity of Ty21a-Induced T Cell Responses to HLA-E Restricted Salmonella Typhi Antigen Presentation.

Human-restricted Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever-a life-threatening disease of great global health significance, particularly in the developing world. Ty21a is an oral live-attenuated vaccine that protects against the development of typhoid disease in part by inducing robust T cell responses, among which multifunctional CD8+ cytotoxic T lymphocytes (CTL) play an important role. Following Ty21a vaccination, a significant component of adult CTL have shown to be targeted to S. Typhi antigen presented by the conserved major histocompatibility complex (MHC) class Ib molecule, human leukocyte antigen-E (HLA-E). S. Typhi challenge studies have shown that baseline, multifunctional HLA-E responsive T cells are associated with protection from, and delayed onset of, typhoid disease. However, despite the overwhelming burden of typhoid fever in school-aged children, and due to limited availability of pediatric samples, incomplete information is available regarding these important HLA-E-restricted responses in children, even though studies have shown that younger children may be less likely to develop protective cell mediated immune (CMI) responses than adults following vaccination. To address this gap, we have studied this phenomenon in depth by using mass cytometry to analyze pediatric and adult T cell responses to HLA-E-restricted S. Typhi antigen presentation, before and after Ty21a vaccination. Herein, we show variable responses in all age strata following vaccination among T effector memory (TEM) and T effector memory CD45RA+ (TEMRA) cells based on conventional gating analysis. However, by utilizing the dimensionality reduction tool tSNE (t-distributed Stochastic Neighbor Embedding), we are able to identify diverse, highly multifunctional gut-homing- TEM and TEMRA clusters of cells which are more abundant in adult and older pediatric participants than in younger children. These findings highlight a potential age-associated maturation of otherwise conserved HLA-E restricted T cell responses. Such insights, coupled with the marked importance of multifunctional T cell responses to combat infection, may better inform future pediatric vaccination strategies against S. Typhi and other infectious diseases.

Differences Between Pediatric and Adult T Cell Responses to In Vitro Staphylococcal Enterotoxin B Stimulation.

Toxic shock syndrome (TSS) is capable of inducing life-threatening fever, rash, and systemic organ failure, though the specific mechanisms behind these symptoms remain poorly understood. Staphylococcal enterotoxin B (SEB) and other superantigens have shown to be important factors in TSS, capable of promoting cross-linking between T cell receptors and major histocompatibility complexes which results in overwhelming T cell activation, proliferation, and cytokine production. The resulting proinflammatory cytokine cascade, often referred to as the "cytokine storm," seems to be critical to the development of disease. Interestingly, clinical studies have shown that children exhibit less severe TSS-associated morbidity than adults, though the mechanism behind this phenomenon has not been addressed. Indeed, despite the fact that most novel antigen exposure occurs early in life, be it from environmentally acquired pathogens or routine vaccination, normal pediatric T cell immune functions remain critically underexplored. This is largely due to difficulty in obtaining enough samples to explore more than a narrow sliver of the cell-mediated immune compartment. To address this limitation, we optimized a T effector (Teff)/circulating T follicular helper (cTFH) cell mass cytometry panel which allowed us to analyze a wide array of T cell populations and effector functions following in vitro SEB stimulation. We show that T cell activation-as measured by CD69 expression-following SEB stimulation is lower in pediatric participants, increasing throughout childhood, and reaching adult levels by around 15 years old. Further, while individual CD4+ effector memory T cell (TEM) effector molecules show limited age-associated differences following SEB stimulation, multifunctional CD4+ TEM are shown to positively correlate with increasing age through adolescence. Individual CD8+ TEM effectors and multifunctional phenotypes also show very strong age-associated increases following SEB stimulation. SEB stimulation has little impact on cTFH activation or functional cellular markers, regardless of age. These results, coupled with the fact that a robust proinflammatory cytokine response seems critical to developing severe TSS, suggest a possible connection between the significantly reduced T cell activation and multifunctional populations following in vitro SEB stimulation in our pediatric participants and clinical observations relating to reduced TSS mortality in children.