Tumor-associated myeloid cells provide critical support for T-ALL.

Despite harboring mutations in oncogenes and tumor suppressors that promote cancer growth, T-cell acute lymphoblastic leukemia (T-ALL) cells require exogenous cells or signals to survive in culture. We previously reported that myeloid cells, particularly dendritic cells, from the thymic tumor microenvironment support the survival and proliferation of primary mouse T-ALL cells in vitro. Thus, we hypothesized that tumor-associated myeloid cells would support T-ALL in vivo. Consistent with this possibility, in vivo depletion of myeloid cells results in a significant reduction in leukemia burden in multiple organs in 2 distinct mouse models of T-ALL and prolongs survival. The impact of the myeloid compartment on T-ALL growth is not dependent on suppression of antitumor T-cell responses. Instead, myeloid cells provide signals that directly support T-ALL cells. Transcriptional profiling, functional assays, and acute in vivo myeloid-depletion experiments identify activation of IGF1R as a critical component of myeloid-mediated T-ALL growth and survival. We identify several myeloid subsets that have the capacity to directly support survival of T-ALL cells. Consistent with mouse models, myeloid cells derived from human peripheral blood monocytes activate IGF1R and directly support survival of primary patient T-ALL cells in vitro. Furthermore, enriched macrophage gene signatures in published clinical samples correlate with inferior outcomes for pediatric T-ALL patients. Collectively, these data reveal that tumor-associated myeloid cells provide signals critical for T-ALL growth in multiple organs in vivo and implicate tumor-associated myeloid cells and associated signals as potential therapeutic targets.

Chemokine-Mediated Choreography of Thymocyte Development and Selection.

As they differentiate, thymocytes encounter spatially restricted cues critical for differentiation and selection of a functional, self-tolerant T cell repertoire. Sequential migration of developing T cells through distinct thymic microenvironments is enforced by the ordered expression of chemokine receptors. Herein, we provide an updated perspective on T cell differentiation through the lens of recent advances that illuminate the dynamics of chemokine-driven thymocyte migration, localization, and interactions with stromal cells. We consider these findings in the context of earlier groundwork exploring the contribution of chemokines to T cell development, recent advances regarding the specificity of chemokine signaling, and novel techniques for evaluating the T cell repertoire. We suggest future research should amalgamate visualization of localized cellular interactions with downstream molecular signals.

Microglia depletion and alcohol: Transcriptome and behavioral profiles.

Alcohol abuse induces changes in microglia morphology and immune function, but whether microglia initiate or simply amplify the harmful effects of alcohol exposure is still a matter of debate. Here, we determine microglia function in acute and voluntary drinking behaviors using a colony-stimulating factor 1 receptor inhibitor (PLX5622). We show that microglia depletion does not alter the sedative or hypnotic effects of acute intoxication. Microglia depletion also does not change the escalation or maintenance of chronic voluntary alcohol consumption. Transcriptomic analysis revealed that although many immune genes have been implicated in alcohol abuse, downregulation of microglia genes does not necessitate changes in alcohol intake. Instead, microglia depletion and chronic alcohol result in compensatory upregulation of alcohol-responsive, reactive astrocyte genes, indicating astrocytes may play a role in regulation of these alcohol behaviors. Taken together, our behavioral and transcriptional data indicate that microglia are not the primary effector cell responsible for regulation of acute and voluntary alcohol behaviors. Because microglia depletion did not regulate acute or voluntary alcohol behaviors, we hypothesized that these doses were insufficient to activate microglia and recruit them to an effector phenotype. Therefore, we used a model of repeated immune activation using polyinosinic:polycytidylic acid (poly(I:C)) to activate microglia. Microglia depletion blocked poly(I:C)-induced escalations in alcohol intake, indicating microglia regulate drinking behaviors with sufficient immune activation. By testing the functional role of microglia in alcohol behaviors, we provide insight into when microglia are causal and when they are consequential for the transition from alcohol use to dependence.

Endogenous dendritic cells from the tumor microenvironment support T-ALL growth via IGF1R activation.

Primary T-cell acute lymphoblastic leukemia (T-ALL) cells require stromal-derived signals to survive. Although many studies have identified cell-intrinsic alterations in signaling pathways that promote T-ALL growth, the identity of endogenous stromal cells and their associated signals in the tumor microenvironment that support T-ALL remains unknown. By examining the thymic tumor microenvironments in multiple murine T-ALL models and primary patient samples, we discovered the emergence of prominent epithelial-free regions, enriched for proliferating tumor cells and dendritic cells (DCs). Systematic evaluation of the functional capacity of tumor-associated stromal cells revealed that myeloid cells, primarily DCs, are necessary and sufficient to support T-ALL survival ex vivo. DCs support T-ALL growth both in primary thymic tumors and at secondary tumor sites. To identify a molecular mechanism by which DCs support T-ALL growth, we first performed gene expression profiling, which revealed up-regulation of platelet-derived growth factor receptor beta (Pdgfrb) and insulin-like growth factor I receptor (Igf1r) on T-ALL cells, with concomitant expression of their ligands by tumor-associated DCs. Both Pdgfrb and Igf1r were activated in ex vivo T-ALL cells, and coculture with tumor-associated, but not normal thymic DCs, sustained IGF1R activation. Furthermore, IGF1R signaling was necessary for DC-mediated T-ALL survival. Collectively, these studies provide the first evidence that endogenous tumor-associated DCs supply signals driving T-ALL growth, and implicate tumor-associated DCs and their mitogenic signals as auspicious therapeutic targets.

EBI2 contributes to the induction of thymic central tolerance in mice by promoting rapid motility of medullary thymocytes.

Maturing thymocytes enter the thymic medulla, where they encounter numerous self-antigens presented by antigen presenting cells (APCs). Those thymocytes that are strongly self-reactive undergo either negative selection or diversion into the regulatory T-cell lineage. Although the majority of the proteome is expressed in the medulla, many self-antigens are expressed by only a minor fraction of medullary APCs; thus, thymocytes must efficiently enter the medulla and scan APCs to ensure central tolerance. Chemokine receptors promote lymphocyte migration, organization within tissues, and interactions with APCs in lymphoid organs. The chemokine receptor EBI2 governs localization of T cells, B cells, and dendritic cells (DCs) during immune responses in secondary lymphoid organs. However, the role of EBI2 in thymocyte development has not been elucidated. Here, we demonstrate that EBI2 is expressed by murine CD4+ single positive (CD4SP) thymocytes and thymic DCs. EBI2 deficiency alters the TCR repertoire, but does not grossly impact thymocyte cellularity or subset distribution. EBI2 deficiency also impairs negative selection of OT-II TCR transgenic thymocytes responding to an endogenous self-antigen. Two-photon imaging revealed that EBI2 deficiency results in reduced migration and impaired medullary accumulation of CD4SP thymocytes. These data identify a role for EBI2 in promoting efficient thymic central tolerance.

Dynein Separately Partners with NDE1 and Dynactin To Orchestrate T Cell Focused Secretion.

Helper and cytotoxic T cells accomplish focused secretion through the movement of vesicles toward the microtubule organizing center (MTOC) and translocation of the MTOC to the target contact site. In this study, using Jurkat cells and OT-I TCR transgenic primary murine CTLs, we show that the dynein-binding proteins nuclear distribution E homolog 1 (NDE1) and dynactin (as represented by p150(Glued)) form mutually exclusive complexes with dynein, exhibit nonoverlapping distributions in target-stimulated cells, and mediate different transport events. When Jurkat cells expressing a dominant negative form of NDE1 (NDE1-enhanced GFP fusion) were activated by Staphylococcus enterotoxin E-coated Raji cells, NDE1 and dynein failed to accumulate at the immunological synapse (IS) and MTOC translocation was inhibited. Knockdown of NDE1 in Jurkat cells or primary mouse CTLs also inhibited MTOC translocation and CTL-mediated killing. In contrast to NDE1, knockdown of p150(Glued), which depleted the alternative dynein/dynactin complex, resulted in impaired accumulation of CTLA4 and granzyme B-containing intracellular vesicles at the IS, whereas MTOC translocation was not affected. Depletion of p150(Glued) in CTLs also inhibited CTL-mediated lysis. We conclude that the NDE1/Lissencephaly 1 and dynactin complexes separately mediate two key components of T cell-focused secretion, namely translocation of the MTOC and lytic granules to the IS, respectively.

Comprehensive methylome map of lineage commitment from haematopoietic progenitors.

Epigenetic modifications must underlie lineage-specific differentiation as terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Haematopoiesis provides a well-defined model to study epigenetic modifications during cell-fate decisions, as multipotent progenitors (MPPs) differentiate into progressively restricted myeloid or lymphoid progenitors. Although DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs, a comprehensive DNA methylation map of haematopoietic progenitors, or of any multipotent/oligopotent lineage, does not exist. Here we examined 4.6 million CpG sites throughout the genome for MPPs, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and thymocyte progenitors (DN1, DN2, DN3). Marked epigenetic plasticity accompanied both lymphoid and myeloid restriction. Myeloid commitment involved less global DNA methylation than lymphoid commitment, supported functionally by myeloid skewing of progenitors following treatment with a DNA methyltransferase inhibitor. Differential DNA methylation correlated with gene expression more strongly at CpG island shores than CpG islands. Many examples of genes and pathways not previously known to be involved in choice between lymphoid/myeloid differentiation have been identified, such as Arl4c and Jdp2. Several transcription factors, including Meis1, were methylated and silenced during differentiation, indicating a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome seems to be important in haematopoietic differentiation. Our results directly demonstrate that modulation of DNA methylation occurs during lineage-specific differentiation and defines a comprehensive map of the methylation and transcriptional changes that accompany myeloid versus lymphoid fate decisions.

Dynamics of BMP signaling in limb bud mesenchyme and polydactyly.

Mutations in the Bone Morphogenetic Protein (BMP) pathway are associated with a range of defects in skeletal formation. Genetic analysis of BMP signaling requirements is complicated by the presence of three partially redundant BMPs that are required for multiple stages of limb development. We generated an inducible allele of a BMP inhibitor, Gremlin, which reduces BMP signaling. We show that BMPs act in a dose and time dependent manner in which early reduction of BMPs result in digit loss, while inhibiting overall BMP signaling between E10.5 and E11.5 allows polydactylous digit formation. During this period, inhibiting BMPs extends the duration of FGF signaling. Sox9 is initially expressed in normal digit ray domains but at reduced levels that correlate with the reduction in BMP signaling. The persistence of elevated FGF signaling likely promotes cell proliferation and survival, inhibiting the activation of Sox9 and secondarily, inhibiting the differentiation of Sox9-expressing chondrocytes. Our results provide new insights into the timing and clarify the mechanisms underlying BMP signaling during digit morphogenesis.

Coactivator-associated arginine methyltransferase 1 regulates fetal hematopoiesis and thymocyte development.

Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein arginine methyltransferase that methylates histones and transcriptional regulators. We previously reported that the absence of CARM1 partially blocks thymocyte differentiation at embryonic day 18.5 (E18.5). In this study, we find that reduced thymopoiesis in Carm1(-/-) mice is due to a defect in the fetal hematopoietic compartment rather than in the thymic stroma. To determine the cellular basis for impaired thymopoiesis, we examined the number and function of fetal liver (FL) and bone marrow cells. Despite markedly reduced cellularity of hematopoietic progenitors in E18.5 bone marrow, the number of long-term hematopoietic stem cells and downstream subsets was not reduced in Carm1(-/-) E14.5 or E18.5 FL. Nevertheless, competitive reconstitution assays revealed a deficit in the ability of Carm1(-/-) FL cells to contribute to hematopoiesis. Furthermore, impaired differentiation of Carm1(-/-) FL cells in a CARM1-sufficient host showed that CARM1 is required cell autonomously in hematopoietic cells. Coculture of Carm1(-/-) FL cells on OP9-DL1 monolayers showed that CARM1 is required for survival of hematopoietic progenitors under conditions that promote differentiation. Taken together, this report demonstrates that CARM1 is a key epigenetic regulator of hematopoiesis that affects multiple lineages at various stages of differentiation.

Lymphoid origin of intrinsically activated plasmacytoid dendritic cells in mice.

We identified a novel mouse plasmacytoid dendritic cell (pDC) lineage derived from the common lymphoid progenitors (CLPs) that is dependent on expression of Bcl11a. These CLP-derived pDCs, which we refer to as 'B-pDCs', have a unique gene expression profile that includes hallmark B cell genes, normally not expressed in conventional pDCs. Despite expressing most classical pDC markers such as SIGLEC-H and PDCA1, B-pDCs lack IFN-α secretion, exhibiting a distinct inflammatory profile. Functionally, B-pDCs induce T cell proliferation more robustly than canonical pDCs following Toll-like receptor 9 (TLR9) engagement. B-pDCs, along with another homogeneous subpopulation of myeloid-derived pDCs, display elevated levels of the cell surface receptor tyrosine kinase AXL, mirroring human AXL+ transitional DCs in function and transcriptional profile. Murine B-pDCs therefore represent a phenotypically and functionally distinct CLP-derived DC lineage specialized in T cell activation and previously not described in mice.

Features of acute COVID-19 associated with post-acute sequelae of SARS-CoV-2 phenotypes: results from the IMPACC study.

Post-acute sequelae of SARS-CoV-2 (PASC) is a significant public health concern. We describe Patient Reported Outcomes (PROs) on 590 participants prospectively assessed from hospital admission for COVID-19 through one year after discharge. Modeling identified 4 PRO clusters based on reported deficits (minimal, physical, mental/cognitive, and multidomain), supporting heterogenous clinical presentations in PASC, with sub-phenotypes associated with female sex and distinctive comorbidities. During the acute phase of disease, a higher respiratory SARS-CoV-2 viral burden and lower Receptor Binding Domain and Spike antibody titers were associated with both the physical predominant and the multidomain deficit clusters. A lower frequency of circulating B lymphocytes by mass cytometry (CyTOF) was observed in the multidomain deficit cluster. Circulating fibroblast growth factor 21 (FGF21) was significantly elevated in the mental/cognitive predominant and the multidomain clusters. Future efforts to link PASC to acute anti-viral host responses may help to better target treatment and prevention of PASC.

Integrin signaling is critical for myeloid-mediated support of T-cell acute lymphoblastic leukemia.

We previously found that T-cell acute lymphoblastic leukemia (T-ALL) requires support from tumor-associated myeloid cells, which activate Insulin Like Growth Factor 1 Receptor (IGF1R) signaling in leukemic blasts. However, IGF1 is not sufficient to sustain T-ALL in vitro, implicating additional myeloid-mediated signals in leukemia progression. Here, we find that T-ALL cells require close contact with myeloid cells to survive. Transcriptional profiling and in vitro assays demonstrate that integrin-mediated cell adhesion activates downstream focal adhesion kinase (FAK)/ proline-rich tyrosine kinase 2 (PYK2), which are required for myeloid-mediated T-ALL support, partly through activation of IGF1R. Blocking integrin ligands or inhibiting FAK/PYK2 signaling diminishes leukemia burden in multiple organs and confers a survival advantage in a mouse model of T-ALL. Inhibiting integrin-mediated adhesion or FAK/PYK2 also reduces survival of primary patient T-ALL cells co-cultured with myeloid cells. Furthermore, elevated integrin pathway gene signatures correlate with higher FAK signaling and myeloid gene signatures and are associated with an inferior prognosis in pediatric T-ALL patients. Together, these findings demonstrate that integrin activation and downstream FAK/PYK2 signaling are important mechanisms underlying myeloid-mediated support of T-ALL progression.

CCR4 and CCR7 differentially regulate thymocyte localization with distinct outcomes for central tolerance.

Central tolerance ensures autoreactive T cells are eliminated or diverted to the regulatory T cell lineage, thus preventing autoimmunity. To undergo central tolerance, thymocytes must enter the medulla to test their T-cell receptors (TCRs) for autoreactivity against the diverse self-antigens displayed by antigen-presenting cells (APCs). While CCR7 is known to promote thymocyte medullary entry and negative selection, our previous studies implicate CCR4 in these processes, raising the question of whether CCR4 and CCR7 play distinct or redundant roles in central tolerance. Here, synchronized positive selection assays, two-photon time-lapse microscopy, and quantification of TCR-signaled apoptotic thymocytes, demonstrate that CCR4 and CCR7 promote medullary accumulation and central tolerance of distinct post-positive selection thymocyte subsets in mice. CCR4 is upregulated within hours of positive selection signaling and promotes medullary entry and clonal deletion of immature post-positive selection thymocytes. In contrast, CCR7 is expressed several days later and is required for medullary localization and negative selection of mature thymocytes. In addition, CCR4 and CCR7 differentially enforce self-tolerance, with CCR4 enforcing tolerance to self-antigens presented by activated APCs, which express CCR4 ligands. Our findings show that CCR7 expression is not synonymous with medullary localization and support a revised model of central tolerance in which CCR4 and CCR7 promote early and late stages of negative selection, respectively, via interactions with distinct APC subsets.

Autoimmune diseases occur when immune cells mistakenly identify the body's own tissues as 'foreign' and attack them. To reduce the risk of this happening, the body has multiple ways of removing self-reactive immune cells, including T cells. One such way, known as central tolerance, occurs in the thymus ­ the organ where T cells develop. In the center of the thymus ­ the medulla ­ specialized cells display fragments of the majority of proteins expressed by healthy cells throughout the body. Developing T cells enter the medulla, where they scan these specialized cells to determine if they recognize the presented protein fragments. If an immature T cell recognizes and binds to these 'self-antigens' too strongly, it is either destroyed, or it develops into a regulatory cell, capable of actively suppressing T cell responses to that self-antigen. This ensures that T cells won't attack healthy cells in the body that make those self-antigens, and therefore, it is important that T cells enter the medulla and carry out this scanning process efficiently. T cells are recruited to the medulla from the outer region of the thymus by chemical signals called chemokines. These signals are recognized by chemokine receptors on T cells, which are expressed at different times during T cell development. Previous work has shown that one of these receptors, called CCR7, guides T cells to the medulla. Although it was thought that CCR7 was solely responsible for this migration, prior work suggests another receptor, CCR4, may also contribute to T cell migration into the medulla and central tolerance. To determine whether CCR7 and CCR4 play the same or different roles in central tolerance, Li, Tipan et al. used a combination of experimental methods, including live imaging of the thymus, to study T cell development in mice. The experiments revealed that CCR4 is expressed first, and this receptor alone guides immature T cells into the medulla and ensures that they are the first to be checked for self-reactivity. In contrast, CCR7 is expressed by more mature developing T cells two to three days later, ensuring they also accumulate within the medulla and become tolerant to self-antigens. Both receptors are required for protection from autoimmunity, with results suggesting that CCR4 and CCR7 promote tolerance against different tissues. Taken together, the findings provide new information about the distinct requirement for CCR4 and CCR7 in guiding immature T cells into the medulla and ensuring central tolerance to diverse tissues. One outstanding question is whether defects in T cells entering the medulla earlier or later alter tolerance to distinct self-antigens and lead to different autoimmune diseases. Future work will also investigate whether these observations hold true in humans, potentially leading to therapies for autoimmune diseases.

The initial age-associated decline in early T-cell progenitors reflects fewer pre-thymic progenitors and altered signals in the bone marrow and thymus microenvironments.

Age-related thymus involution results in decreased T-cell production, contributing to increased susceptibility to pathogens and reduced vaccine responsiveness. Elucidating mechanisms underlying thymus involution will inform strategies to restore thymopoiesis with age. The thymus is colonized by circulating bone marrow (BM)-derived thymus seeding progenitors (TSPs) that differentiate into early T-cell progenitors (ETPs). We find that ETP cellularity declines as early as 3 months (3MO) of age in mice. This initial ETP reduction could reflect changes in thymic stromal niches and/or pre-thymic progenitors. Using a multicongenic progenitor transfer approach, we demonstrate that the number of functional TSP/ETP niches does not diminish with age. Instead, the number of pre-thymic lymphoid progenitors in the BM and blood is substantially reduced by 3MO, although their intrinsic ability to seed and differentiate in the thymus is maintained. Additionally, Notch signaling in BM lymphoid progenitors and in ETPs diminishes by 3MO, suggesting reduced niche quality in the BM and thymus contribute to the early decline in ETPs. Together, these findings indicate that diminished BM lymphopoiesis and thymic stromal support contribute to an initial reduction in ETPs in young adulthood, setting the stage for progressive age-associated thymus involution.

SARS-CoV-2 Humoral Immune Responses in Convalescent Individuals Over 12 Months Reveal Severity-Dependent Antibody Dynamics.

Background: Understanding the kinetics and longevity of antibody responses to SARS-CoV-2 is critical to informing strategies toward reducing Coronavirus disease 2019 (COVID-19) reinfections, and improving vaccination and therapy approaches. Methods: We evaluated antibody titers against SARS-CoV-2 nucleocapsid (N), spike (S), and receptor binding domain (RBD) of spike in 98 convalescent participants who experienced asymptomatic, mild, moderate or severe COVID-19 disease and in 17 non-vaccinated, non-infected controls, using four different antibody assays. Participants were sampled longitudinally at 1, 3, 6, and 12 months post-SARS-CoV-2 positive PCR test. Findings: Increasing acute COVID-19 disease severity correlated with higher anti-N and anti-RBD antibody titers throughout 12 months post-infection. Anti-N and anti-RBD titers declined over time in all participants, with the exception of increased anti-RBD titers post-vaccination, and the decay rates were faster in hospitalized compared to non-hospitalized participants. <50% of participants retained anti-N titers above control levels at 12 months, with non-hospitalized participants falling below control levels sooner. Nearly all hospitalized and non-hospitalized participants maintained anti-RBD titers above controls for up to 12 months, suggesting longevity of protection against severe reinfections. Nonetheless, by 6 months, few participants retained >50% of their 1-month anti-N or anti-RBD titers. Vaccine-induced increases in anti-RBD titers were greater in non-hospitalized relative to hospitalized participants. Early convalescent antibody titers correlated with age, but no association was observed between Post-Acute Sequelae of SARS-CoV-2 infection (PASC) status or acute steroid treatment and convalescent antibody titers. Interpretation: Hospitalized participants developed higher anti-SARS-CoV-2 antibody titers relative to non-hospitalized participants, a difference that persisted throughout 12 months, despite the faster decline in titers in hospitalized participants. In both groups, while anti-N titers fell below control levels for at least half of the participants, anti-RBD titers remained above control levels for almost all participants over 12 months, demonstrating generation of long-lived antibody responses known to correlate with protection from severe disease across COVID-19 severities. Overall, our findings contribute to the evolving understanding of COVID-19 antibody dynamics. Funding: Austin Public Health, NIAAA, Babson Diagnostics, Dell Medical School Startup.

Integrated longitudinal multi-omics study identifies immune programs associated with COVID-19 severity and mortality in 1152 hospitalized participants.

Hospitalized COVID-19 patients exhibit diverse clinical outcomes, with some individuals diverging over time even though their initial disease severity appears similar. A systematic evaluation of molecular and cellular profiles over the full disease course can link immune programs and their coordination with progression heterogeneity. In this study, we carried out deep immunophenotyping and conducted longitudinal multi-omics modeling integrating ten distinct assays on a total of 1,152 IMPACC participants and identified several immune cascades that were significant drivers of differential clinical outcomes. Increasing disease severity was driven by a temporal pattern that began with the early upregulation of immunosuppressive metabolites and then elevated levels of inflammatory cytokines, signatures of coagulation, NETosis, and T-cell functional dysregulation. A second immune cascade, predictive of 28-day mortality among critically ill patients, was characterized by reduced total plasma immunoglobulins and B cells, as well as dysregulated IFN responsiveness. We demonstrated that the balance disruption between IFN-stimulated genes and IFN inhibitors is a crucial biomarker of COVID-19 mortality, potentially contributing to the failure of viral clearance in patients with fatal illness. Our longitudinal multi-omics profiling study revealed novel temporal coordination across diverse omics that potentially explain disease progression, providing insights that inform the targeted development of therapies for hospitalized COVID-19 patients, especially those critically ill.

Central tolerance is impaired in the middle-aged thymic environment.

One of the earliest hallmarks of immune aging is thymus involution, which not only reduces the number of newly generated and exported T cells, but also alters the composition and organization of the thymus microenvironment. Thymic T-cell export continues into adulthood, yet the impact of thymus involution on the quality of newly generated T-cell clones is not well established. Notably, the number and proportion of medullary thymic epithelial cells (mTECs) and expression of tissue-restricted antigens (TRAs) decline with age, suggesting the involuting thymus may not promote efficient central tolerance. Here, we demonstrate that the middle-aged thymic environment does not support rapid motility of medullary thymocytes, potentially diminishing their ability to scan antigen presenting cells (APCs) that display the diverse self-antigens that induce central tolerance. Consistent with this possibility, thymic slice assays reveal that the middle-aged thymic environment does not support efficient negative selection or regulatory T-cell (Treg) induction of thymocytes responsive to either TRAs or ubiquitous self-antigens. This decline in central tolerance is not universal, but instead impacts lower-avidity self-antigens that are either less abundant or bind to TCRs with moderate affinities. Additionally, the decline in thymic tolerance by middle age is accompanied by both a reduction in mTECs and hematopoietic APC subsets that cooperate to drive central tolerance. Thus, age-associated changes in the thymic environment result in impaired central tolerance against moderate-avidity self-antigens, potentially resulting in export of increasingly autoreactive naive T cells, with a deficit of Treg counterparts by middle age.

Longitudinal COVID-19 immune trajectories in patients with neurological autoimmunity on anti-CD20 therapy.

BACKGROUND AND OBJECTIVES: During the COVID-19 pandemic, B cell depleting therapies pose a clinical concern for patients with neuroimmune conditions, as patients may not mount a sufficient immune response to SARS-CoV-2 infection and vaccinations. Studies to-date have reported conflicting results on the degree of antibody production post-SARS-CoV-2 infection and vaccinations in B cell depleted patients, focusing primarily on short-term immune profiling. Our objective was to follow longitudinal immune responses in COVID-19 B cell depleted patients with neuroimmune disorders post-COVID-19 and SARS-CoV-2-vaccination. METHODS: CD20 B cell depleted autoimmune patients and age/sex-matched controls positive for SARS-CoV-2 were recruited at Dell Medical School, UT Austin between 2020 and 2021, followed prospectively for 12 months and evaluated at multiple time points for spike S1 receptor binding domain (RBD) antibody titers, B and T cell composition, and frequency of T cells specific for SARS-CoV-2 antigens. RESULTS: Immune responses post-SARS-CoV-2 infection and vaccination were evaluated in a cohort of COVID-19 B cell depleted neuroimmune patients (n = 5), COVID-19 non-B cell depleted autoimmune patients (n = 15), COVID-19 immunocompetent patients (n = 117), and healthy controls (n = 6) for a total of 259 samples in 137 participants. 4/5 B cell-depleted patients developed detectable anti-spike RBD antibodies, which were boosted by vaccination in 2 patients. While spike RBD antibodies were associated with presence of CD20+ B cells, very few B cells were required. In contrast, patients whose B cell compartment primarily consisted of CD19+CD20- Bcells during acute COVID-19 disease or vaccination did not seroconvert. Interestingly, circulating Bcells in B cell depleted patients were significantly CD38high with co-expression of CD24 and CD27, indicating that B cell depletion may impact B cell activation patterns. Additionally, all B cell depleted patients mounted a sustained T cell response to SARS-CoV-2 antigens, regardless of seroconversion. Specifically, all patients developed naïve, central memory, effector memory, and effector memory RA+ T cells, suggesting intact T cell memory conversion in B cell depleted patients compared to controls. DISCUSSION: We present the longest COVID-19 immune profiling analysis to date in B cell depleted patients, demonstrating that both humoral and cellular immune responses can be generated and sustained up to 12 months post SARS-CoV-2 infection and vaccination. Notably, failure to establish humoral immunity did not result in severe disease. We also highlight specific T and B cell signatures that could be used as clinical biomarkers to advise patients on timing of SARS-CoV-2 vaccinations.

Phenotypes of disease severity in a cohort of hospitalized COVID-19 patients: Results from the IMPACC study.

BACKGROUND: Better understanding of the association between characteristics of patients hospitalized with coronavirus disease 2019 (COVID-19) and outcome is needed to further improve upon patient management. METHODS: Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC) is a prospective, observational study of 1164 patients from 20 hospitals across the United States. Disease severity was assessed using a 7-point ordinal scale based on degree of respiratory illness. Patients were prospectively surveyed for 1 year after discharge for post-acute sequalae of COVID-19 (PASC) through quarterly surveys. Demographics, comorbidities, radiographic findings, clinical laboratory values, SARS-CoV-2 PCR and serology were captured over a 28-day period. Multivariable logistic regression was performed. FINDINGS: The median age was 59 years (interquartile range [IQR] 20); 711 (61%) were men; overall mortality was 14%, and 228 (20%) required invasive mechanical ventilation. Unsupervised clustering of ordinal score over time revealed distinct disease course trajectories. Risk factors associated with prolonged hospitalization or death by day 28 included age ≥ 65 years (odds ratio [OR], 2.01; 95% CI 1.28-3.17), Hispanic ethnicity (OR, 1.71; 95% CI 1.13-2.57), elevated baseline creatinine (OR 2.80; 95% CI 1.63- 4.80) or troponin (OR 1.89; 95% 1.03-3.47), baseline lymphopenia (OR 2.19; 95% CI 1.61-2.97), presence of infiltrate by chest imaging (OR 3.16; 95% CI 1.96-5.10), and high SARS-CoV2 viral load (OR 1.53; 95% CI 1.17-2.00). Fatal cases had the lowest ratio of SARS-CoV-2 antibody to viral load levels compared to other trajectories over time (p=0.001). 589 survivors (51%) completed at least one survey at follow-up with 305 (52%) having at least one symptom consistent with PASC, most commonly dyspnea (56% among symptomatic patients). Female sex was the only associated risk factor for PASC. INTERPRETATION: Integration of PCR cycle threshold, and antibody values with demographics, comorbidities, and laboratory/radiographic findings identified risk factors for 28-day outcome severity, though only female sex was associated with PASC. Longitudinal clinical phenotyping offers important insights, and provides a framework for immunophenotyping for acute and long COVID-19. FUNDING: NIH.

Age-Related Changes in Thymic Central Tolerance.

Thymic epithelial cells (TECs) and hematopoietic antigen presenting cells (HAPCs) in the thymus microenvironment provide essential signals to self-reactive thymocytes that induce either negative selection or generation of regulatory T cells (Treg), both of which are required to establish and maintain central tolerance throughout life. HAPCs and TECs are comprised of multiple subsets that play distinct and overlapping roles in central tolerance. Changes that occur in the composition and function of TEC and HAPC subsets across the lifespan have potential consequences for central tolerance. In keeping with this possibility, there are age-associated changes in the cellular composition and function of T cells and Treg. This review summarizes changes in T cell and Treg function during the perinatal to adult transition and in the course of normal aging, and relates these changes to age-associated alterations in thymic HAPC and TEC subsets.