Publications by authors named "Michael R Betts"

132 Publications

Longitudinal Analysis Reveals Distinct Antibody and Memory B Cell Responses in SARS-CoV2 Naïve and Recovered Individuals Following mRNA Vaccination.

medRxiv 2021 Mar 6. Epub 2021 Mar 6.

Novel mRNA vaccines for SARS-CoV2 have been authorized for emergency use and are currently being administered to millions of individuals worldwide. Despite their efficacy in clinical trials, there is limited data on vaccine-induced immune responses in individuals with a prior SARS-CoV2 infection compared to SARS-CoV2 naïve subjects. Moreover, how mRNA vaccines impact the development of antibodies as well as memory B cells in COVID-19 experienced versus COVID-19 naïve subjects remains poorly understood. In this study, we evaluated antibody responses and antigen-specific memory B cell responses over time in 33 SARS-CoV2 naïve and 11 SARS-CoV2 recovered subjects. mRNA vaccination induced significant antibody and memory B cell responses against full-length SARS-CoV2 spike protein and the spike receptor binding domain (RBD). SARS-CoV2 naïve individuals benefitted from both doses of mRNA vaccine with additional increases in antibodies and memory B cells following booster immunization. In contrast, SARS-CoV2 recovered individuals had a significant immune response after the first dose with no increase in circulating antibodies or antigen-specific memory B cells after the second dose. Moreover, the magnitude of the memory B cell response induced by vaccination was lower in older individuals, revealing an age-dependence to mRNA vaccine-induced B cell memory. Side effects also tended to associate with post-boost antibody levels, but not with post-boost memory B cells, suggesting that side effect severity may be a surrogate of short-term antibody responses. The frequency of pre-vaccine antigen-specific memory B cells in SARS-CoV2 recovered individuals strongly correlated with post-vaccine antibody levels, supporting a key role for memory B cells in humoral recall responses to SARS-CoV2. This observation may have relevance for future booster vaccines and for responses to viral variants that partially escape pre-existing antibodies and require new humoral responses to be generated from memory B cells. Finally, post-boost antibody levels were not correlated with post-boost memory responses in SARS-CoV2 naïve individuals, indicating that short-term antibody levels and memory B cells are complementary immunological endpoints that should be examined in tandem when evaluating vaccine response. Together, our data provide evidence of both serological response and immunological memory following mRNA vaccination that is distinct based on prior SARS-CoV2 exposure. These findings may inform vaccine distribution in a resource-limited setting.

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http://dx.doi.org/10.1101/2021.03.03.21252872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941668PMC
March 2021

Deep immune profiling of MIS-C demonstrates marked but transient immune activation compared to adult and pediatric COVID-19.

Sci Immunol 2021 03;6(57)

Institute for Immunology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.

Pediatric COVID-19 following SARS-CoV-2 infection is associated with fewer hospitalizations and often milder disease than in adults. A subset of children, however, present with Multisystem Inflammatory Syndrome in Children (MIS-C) that can lead to vascular complications and shock, but rarely death. The immune features of MIS-C compared to pediatric COVID-19 or adult disease remain poorly understood. We analyzed peripheral blood immune responses in hospitalized SARS-CoV-2 infected pediatric patients (pediatric COVID-19) and patients with MIS-C. MIS-C patients had patterns of T cell-biased lymphopenia and T cell activation similar to severely ill adults, and all patients with MIS-C had SARS-CoV-2 spike-specific antibodies at admission. A distinct feature of MIS-C patients was robust activation of vascular patrolling CX3CR1+ CD8+ T cells that correlated with the use of vasoactive medication. Finally, whereas pediatric COVID-19 patients with acute respiratory distress syndrome (ARDS) had sustained immune activation, MIS-C patients displayed clinical improvement over time, concomitant with decreasing immune activation. Thus, non-MIS-C versus MIS-C SARS-CoV-2 associated illnesses are characterized by divergent immune signatures that are temporally distinct from one another and implicate CD8+ T cells in the clinical presentation and trajectory of MIS-C.
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http://dx.doi.org/10.1126/sciimmunol.abf7570DOI Listing
March 2021

Seasonal human coronavirus antibodies are boosted upon SARS-CoV-2 infection but not associated with protection.

Cell 2021 04 9;184(7):1858-1864.e10. Epub 2021 Feb 9.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread within the human population. Although SARS-CoV-2 is a novel coronavirus, most humans had been previously exposed to other antigenically distinct common seasonal human coronaviruses (hCoVs) before the coronavirus disease 2019 (COVID-19) pandemic. Here, we quantified levels of SARS-CoV-2-reactive antibodies and hCoV-reactive antibodies in serum samples collected from 431 humans before the COVID-19 pandemic. We then quantified pre-pandemic antibody levels in serum from a separate cohort of 251 individuals who became PCR-confirmed infected with SARS-CoV-2. Finally, we longitudinally measured hCoV and SARS-CoV-2 antibodies in the serum of hospitalized COVID-19 patients. Our studies indicate that most individuals possessed hCoV-reactive antibodies before the COVID-19 pandemic. We determined that ∼20% of these individuals possessed non-neutralizing antibodies that cross-reacted with SARS-CoV-2 spike and nucleocapsid proteins. These antibodies were not associated with protection against SARS-CoV-2 infections or hospitalizations, but they were boosted upon SARS-CoV-2 infection.
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http://dx.doi.org/10.1016/j.cell.2021.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7871851PMC
April 2021

TCR/BCR dual-expressing cells and their associated public BCR clonotype are not enriched in type 1 diabetes.

Cell 2021 Feb;184(3):827-839.e14

Department of Microbiology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19104, USA; Institute for Immunology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19104, USA. Electronic address:

Ahmed and colleagues recently described a novel hybrid lymphocyte expressing both a B and T cell receptor, termed double expresser (DE) cells. DE cells in blood of type 1 diabetes (T1D) subjects were present at increased numbers and enriched for a public B cell clonotype. Here, we attempted to reproduce these findings. While we could identify DE cells by flow cytometry, we found no association between DE cell frequency and T1D status. We were unable to identify the reported public B cell clone, or any similar clone, in bulk B cells or sorted DE cells from T1D subjects or controls. We also did not observe increased usage of the public clone VH or DH genes in B cells or in sorted DE cells. Taken together, our findings suggest that DE cells and their alleged public clonotype are not enriched in T1D. This Matters Arising paper is in response to Ahmed et al. (2019), published in Cell. See also the response by Ahmed et al. (2021), published in this issue.
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http://dx.doi.org/10.1016/j.cell.2020.11.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016147PMC
February 2021

Norovirus-Specific CD8 T Cell Responses in Human Blood and Tissues.

Cell Mol Gastroenterol Hepatol 2021 Jan 11;11(5):1267-1289. Epub 2021 Jan 11.

Department of Medicine, Division of Gastroenterology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; Institute for Immunology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania. Electronic address:

Background & Aims: Noroviruses (NoVs) are the leading cause of acute gastroenteritis worldwide and are associated with significant morbidity and mortality. Moreover, an asymptomatic carrier state can persist following acute infection, promoting NoV spread and evolution. Thus, defining immune correlates of NoV protection and persistence is needed to guide the development of future vaccines and limit viral spread. Whereas antibody responses following NoV infection or vaccination have been studied extensively, cellular immunity has received less attention. Data from the mouse NoV model suggest that T cells are critical for preventing persistence and achieving viral clearance, but little is known about NoV-specific T-cell immunity in humans, particularly at mucosal sites.

Methods: We screened peripheral blood mononuclear cells from 3 volunteers with an overlapping NoV peptide library. We then used HLA-peptide tetramers to track virus-specific CD8 T cells in peripheral, lymphoid, and intestinal tissues. Tetramer cells were further characterized using markers for cellular trafficking, exhaustion, cytotoxicity, and proliferation.

Results: We defined 7 HLA-restricted immunodominant class I epitopes that were highly conserved across pandemic strains from genogroup II.4. NoV-specific CD8 T cells with central, effector, or tissue-resident memory phenotypes were present at all sites and were especially abundant in the intestinal lamina propria. The properties and differentiation states of tetramer cells varied across donors and epitopes.

Conclusions: Our findings are an important step toward defining the breadth, distribution, and properties of human NoV T-cell immunity. Moreover, the molecular tools we have developed can be used to evaluate future vaccines and engineer novel cellular therapeutics.
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http://dx.doi.org/10.1016/j.jcmgh.2020.12.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010716PMC
January 2021

Immunogenicity of an AAV-based, room-temperature stable, single dose COVID-19 vaccine in mice and non-human primates.

bioRxiv 2021 Jan 5. Epub 2021 Jan 5.

The SARS-CoV-2 pandemic has affected more than 70 million people worldwide and resulted in over 1.5 million deaths. A broad deployment of effective immunization campaigns to achieve population immunity at global scale will depend on the biological and logistical attributes of the vaccine. Here, two adeno-associated viral (AAV)-based vaccine candidates demonstrate potent immunogenicity in mouse and nonhuman primates following a single injection. Peak neutralizing antibody titers remain sustained at 5 months and are complemented by functional memory T-cells responses. The AAVrh32.33 capsid of the AAVCOVID vaccine is an engineered AAV to which no relevant pre-existing immunity exists in humans. Moreover, the vaccine is stable at room temperature for at least one month and is produced at high yields using established commercial manufacturing processes in the gene therapy industry. Thus, this methodology holds as a very promising single dose, thermostable vaccine platform well-suited to address emerging pathogens on a global scale.
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http://dx.doi.org/10.1101/2021.01.05.422952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7805441PMC
January 2021

Phenotypic Characterization of SLe+ and CLA+ CD4+ T Cells.

STAR Protoc 2020 Dec 29;1(3):100154. Epub 2020 Oct 29.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Recent advances in high-resolution multiparametric flow cytometry enable ever deeper analysis of human lymphocyte subsets that require rigorous methodology development and optimization. Here, we detail methods to characterize glycosylated Sialyl-Lewis (SLe)- or cutaneous lymphocyte-associated antigen (CLA)-expressing CD4+ T cells using two separate multiparametric flow cytometry panels enabling the identification of memory subsets, Th subsets, and expression of diverse activation markers and chemokine receptors. The proposed protocol allows optimal resolution of the measured parameters while minimizing background in a 25-parameter experiment. For complete details on the use and execution of this protocol, please refer to Colomb et al. (2020).
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http://dx.doi.org/10.1016/j.xpro.2020.100154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757334PMC
December 2020

The Identity of Human Tissue-Emigrant CD8 T Cells.

Cell 2020 Dec 10;183(7):1946-1961.e15. Epub 2020 Dec 10.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Electronic address:

Lymphocyte migration is essential for adaptive immune surveillance. However, our current understanding of this process is rudimentary, because most human studies have been restricted to immunological analyses of blood and various tissues. To address this knowledge gap, we used an integrated approach to characterize tissue-emigrant lineages in thoracic duct lymph (TDL). The most prevalent immune cells in human and non-human primate efferent lymph were T cells. Cytolytic CD8 T cell subsets with effector-like epigenetic and transcriptional signatures were clonotypically skewed and selectively confined to the intravascular circulation, whereas non-cytolytic CD8 T cell subsets with stem-like epigenetic and transcriptional signatures predominated in tissues and TDL. Moreover, these anatomically distinct gene expression profiles were recapitulated within individual clonotypes, suggesting parallel differentiation programs independent of the expressed antigen receptor. Our collective dataset provides an atlas of the migratory immune system and defines the nature of tissue-emigrant CD8 T cells that recirculate via TDL.
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http://dx.doi.org/10.1016/j.cell.2020.11.019DOI Listing
December 2020

Deep Immune Profiling of MIS-C demonstrates marked but transient immune activation compared to adult and pediatric COVID-19.

medRxiv 2020 Sep 27. Epub 2020 Sep 27.

Pediatric COVID-19 following SARS-CoV-2 infection is associated with fewer hospitalizations and often milder disease than in adults. A subset of children, however, present with Multisystem Inflammatory Syndrome in Children (MIS-C) that can lead to vascular complications and shock, but rarely death. The immune features of MIS-C compared to pediatric COVID-19 or adult disease remain poorly understood. We analyzed peripheral blood immune responses in hospitalized SARS-CoV-2 infected pediatric patients (pediatric COVID-19) and patients with MIS-C. MIS-C patients had patterns of T cell-biased lymphopenia and T cell activation similar to severely ill adults, and all patients with MIS-C had SARS-CoV-2 spike-specific antibodies at admission. A distinct feature of MIS-C patients was robust activation of vascular patrolling CX3CR1+ CD8 T cells that correlated with use of vasoactive medication. Finally, whereas pediatric COVID-19 patients with acute respiratory distress syndrome (ARDS) had sustained immune activation, MIS-C patients displayed clinical improvement over time, concomitant with decreasing immune activation. Thus, non-MIS-C versus MIS-C SARS-CoV-2 associated illnesses are characterized by divergent immune signatures that are temporally distinct and implicate CD8 T cells in clinical presentation and trajectory of MIS-C.
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http://dx.doi.org/10.1101/2020.09.25.20201863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523167PMC
September 2020

Assessment of HIV-1 integration in tissues and subsets across infection stages.

JCI Insight 2020 10 15;5(20). Epub 2020 Oct 15.

Department of Microbiology and.

The integration of HIV DNA into the host genome contributes to lifelong infection in most individuals. Few studies have examined integration in lymphoid tissue, where HIV predominantly persists before and after antiretroviral treatment (ART). Of particular interest is whether integration site distributions differ between infection stages with paired blood and tissue comparisons. Here, we profiled HIV integration site distributions in sorted memory, tissue-resident, and/or follicular helper CD4+ T cell subsets from paired blood and lymphoid tissue samples from acute, chronic, and ART-treated individuals. We observed minor differences in the frequency of nonintronic and nondistal intergenic sites, varying with tissue and residency phenotypes during ART. Genomic and epigenetic annotations were generally similar. Clonal expansion of cells marked by identical integration sites was detected, with increased detection in chronic and ART-treated individuals. However, overlap between or within CD4+ T cell subsets or tissue compartments was only observed in 8 unique sites of the 3540 sites studied. Together, these findings suggest that shared integration sites between blood and tissue may, depending on the tissue site, be the exception rather than the rule and indicate that additional studies are necessary to fully understand the heterogeneity of tissue-sequestered HIV reservoirs.
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http://dx.doi.org/10.1172/jci.insight.139783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605534PMC
October 2020

Recommendations for measuring HIV reservoir size in cure-directed clinical trials.

Nat Med 2020 09 7;26(9):1339-1350. Epub 2020 Sep 7.

The Wistar Institute, Philadelphia, PA, USA.

Therapeutic strategies are being clinically tested either to eradicate latent HIV reservoirs or to achieve virologic control in the absence of antiretroviral therapy. Attaining this goal will require a consensus on how best to measure the numbers of persistently infected cells with the potential to cause viral rebound after antiretroviral-therapy cessation in assessing the results of cure-directed strategies in vivo. Current measurements assess various aspects of the HIV provirus and its functionality and produce divergent results. Here, we provide recommendations from the BEAT-HIV Martin Delaney Collaboratory on which viral measurements should be prioritized in HIV-cure-directed clinical trials.
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http://dx.doi.org/10.1038/s41591-020-1022-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7703694PMC
September 2020

Interferon-α alters host glycosylation machinery during treated HIV infection.

EBioMedicine 2020 Sep 19;59:102945. Epub 2020 Aug 19.

The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA. Electronic address:

Background: A comprehensive understanding of host factors modulated by the antiviral cytokine interferon-α (IFNα) is imperative for harnessing its beneficial effects while avoiding its detrimental side-effects during HIV infection. Cytokines modulate host glycosylation which plays a critical role in mediating immunological functions. However, the impact of IFNα on host glycosylation has never been characterized.

Methods: We assessed the impact of pegylated IFNα2a on IgG glycome, as well as CD8 T and NK cell-surface glycomes, of 18 HIV-infected individuals on suppressive antiretroviral therapy. We linked these glycomic signatures to changes in inflammation, CD8 T and NK cell phenotypes, and HIV DNA.

Findings: We identified significant interactions that support a model in which a) IFNα increases the proportion of pro-inflammatory, bisecting GlcNAc glycans (known to enhance FcγR binding) within the IgG glycome, which in turn b) increases inflammation, which c) leads to poor CD8 T cell phenotypes and poor IFNα-mediated reduction of HIV DNA. Examining cell-surface glycomes, IFNα increases levels of the immunosuppressive GalNAc-containing glycans (T/Tn antigens) on CD8 T cells. This induction is associated with lower HIV-gag-specific CD8 T cell functions. Last, IFNα increases levels of fucose on NK cells. This induction is associated with higher NK functions upon K562 stimulation.

Interpretation: IFNα causes host glycomic alterations that are known to modulate immunological responses. These alterations are associated with both detrimental and beneficial consequences of IFNα. Manipulating host glycomic interactions may represent a strategy for enhancing the positive effects of IFNα while avoiding its detrimental side-effects.

Funding: NIH grants R21AI143385, U01AI110434.
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http://dx.doi.org/10.1016/j.ebiom.2020.102945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452630PMC
September 2020

Sialyl-Lewis Glycoantigen Is Enriched on Cells with Persistent HIV Transcription during Therapy.

Cell Rep 2020 08;32(5):107991

The Wistar Institute, Philadelphia, PA 19104, USA; Penn Center for AIDS Research (Penn CFAR), University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

A comprehensive understanding of the phenotype of persistent HIV-infected cells, transcriptionally active and/or transcriptionally inactive, is imperative for developing a cure. The relevance of cell-surface glycosylation to HIV persistence has never been explored. We characterize the relationship between cell-surface glycomic signatures and persistent HIV transcription in vivo. We find that the cell surface of CD4 T cells actively transcribing HIV, despite suppressive therapy, harbors high levels of fucosylated carbohydrate ligands, including the cell extravasation mediator Sialyl-Lewis (SLe), compared with HIV-infected transcriptionally inactive cells. These high levels of SLe are induced by HIV transcription in vitro and are maintained after therapy in vivo. Cells with high-SLe are enriched with markers associated with HIV susceptibility, signaling pathways that drive HIV transcription, and pathways involved in leukocyte extravasation. We describe a glycomic feature of HIV-infected transcriptionally active cells that not only differentiates them from their transcriptionally inactive counterparts but also may affect their trafficking abilities.
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http://dx.doi.org/10.1016/j.celrep.2020.107991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432956PMC
August 2020

Regional Brain Recovery from Acute Synaptic Injury in Simian Immunodeficiency Virus-Infected Rhesus Macaques Associates with Heme Oxygenase Isoform Expression.

J Virol 2020 09 15;94(19). Epub 2020 Sep 15.

Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

Brain injury occurs within days in simian immunodeficiency virus (SIV) or human immunodeficiency virus (HIV) infection, and some recovery may occur within weeks. Inflammation and oxidative stress associate with such injury, but what drives recovery is unknown. Chronic HIV infection associates with reduced brain frontal cortex expression of the antioxidant/anti-inflammatory enzyme heme oxygenase-1 (HO-1) and increased neuroinflammation in individuals with cognitive impairment. We hypothesized that acute regional brain injury and recovery associate with differences in regional brain HO-1 expression. Using SIV-infected rhesus macaques, we analyzed multiple brain regions through acute and chronic infection (90 days postinfection [dpi]) and quantified viral (SIV RNA), synaptic (PSD-95; synaptophysin), axonal (neurofilament/neurofilament light chain [NFL]), inflammatory, and antioxidant (enzymes, including heme oxygenase isoforms [HO-1, HO-2]) markers. PSD-95 was reduced in the brainstem, basal ganglia, neocortex, and cerebellum within 13 dpi, indicating acute synaptic injury throughout the brain. All areas except the brainstem recovered. Unchanged NFL was consistent with no acute axonal injury. SIV RNA expression was highest in the brainstem throughout infection, and it associated with neuroinflammation. Surprisingly, during the synaptic injury and recovery phases, HO-2, and not HO-1, progressively decreased in the brainstem. Thus, acute SIV synaptic injury occurs throughout the brain, with spontaneous recovery in regions other than the brainstem. Within the brainstem, the high SIV load and inflammation, along with reduction of HO-2, may impair recovery. In other brain regions, stable HO-2 expression, with or without increasing HO-1, may promote recovery. Our data support roles for heme oxygenase isoforms in modulating recovery from synaptic injury in SIV infection and suggest their therapeutic targeting for promoting neuronal recovery. Brain injury induced by acute simian (or human) immunodeficiency virus infection may persist or spontaneously resolve in different brain regions. Identifying the host factor(s) that promotes spontaneous recovery from such injury may reveal targets for therapeutic drug strategies for promoting recovery from acute neuronal injury. The gradual recovery from such injury observed in many, but not all, brain regions in the rhesus macaque model is consistent with the possible existence of a therapeutic window of opportunity for intervening to promote recovery, even in those regions not showing spontaneous recovery. In persons living with human immunodeficiency virus infection, such neuroprotective treatments could ultimately be considered as adjuncts to the initiation of antiretroviral drug therapy.
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http://dx.doi.org/10.1128/JVI.01102-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7495379PMC
September 2020

Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications.

Science 2020 09 15;369(6508). Epub 2020 Jul 15.

Institute for Immunology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.

Coronavirus disease 2019 (COVID-19) is currently a global pandemic, but human immune responses to the virus remain poorly understood. We used high-dimensional cytometry to analyze 125 COVID-19 patients and compare them with recovered and healthy individuals. Integrated analysis of ~200 immune and ~50 clinical features revealed activation of T cell and B cell subsets in a proportion of patients. A subgroup of patients had T cell activation characteristic of acute viral infection and plasmablast responses reaching >30% of circulating B cells. However, another subgroup had lymphocyte activation comparable with that in uninfected individuals. Stable versus dynamic immunological signatures were identified and linked to trajectories of disease severity change. Our analyses identified three immunotypes associated with poor clinical trajectories versus improving health. These immunotypes may have implications for the design of therapeutics and vaccines for COVID-19.
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http://dx.doi.org/10.1126/science.abc8511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402624PMC
September 2020

Comprehensive mapping of immune perturbations associated with severe COVID-19.

Sci Immunol 2020 07;5(49)

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Although critical illness has been associated with SARS-CoV-2-induced hyperinflammation, the immune correlates of severe COVID-19 remain unclear. Here, we comprehensively analyzed peripheral blood immune perturbations in 42 SARS-CoV-2 infected and recovered individuals. We identified extensive induction and activation of multiple immune lineages, including T cell activation, oligoclonal plasmablast expansion, and Fc and trafficking receptor modulation on innate lymphocytes and granulocytes, that distinguished severe COVID-19 cases from healthy donors or SARS-CoV-2-recovered or moderate severity patients. We found the neutrophil to lymphocyte ratio to be a prognostic biomarker of disease severity and organ failure. Our findings demonstrate broad innate and adaptive leukocyte perturbations that distinguish dysregulated host responses in severe SARS-CoV-2 infection and warrant therapeutic investigation.
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http://dx.doi.org/10.1126/sciimmunol.abd7114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402634PMC
July 2020

TOX is expressed by exhausted and polyfunctional human effector memory CD8 T cells.

Sci Immunol 2020 07;5(49)

Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Stockholm, Sweden.

CD8 T cell exhaustion is a hallmark of many cancers and chronic infections. In mice, T cell factor 1 (TCF-1) maintains exhausted CD8 T cell responses, whereas thymocyte selection-associated HMG box (TOX) is required for the epigenetic remodeling and survival of exhausted CD8 T cells. However, it has remained unclear to what extent these transcription factors play analogous roles in humans. In this study, we mapped the expression of TOX and TCF-1 as a function of differentiation and specificity in the human CD8 T cell landscape. Here, we demonstrate that circulating TOX CD8 T cells exist in most humans, but that TOX is not exclusively associated with exhaustion. Effector memory CD8 T cells generally expressed TOX, whereas naive and early-differentiated memory CD8 T cells generally expressed TCF-1. Cytolytic gene and protein expression signatures were also defined by the expression of TOX. In the context of a relentless immune challenge, exhausted HIV-specific CD8 T cells commonly expressed TOX, often in clusters with various activation markers and inhibitory receptors, and expressed less TCF-1. However, polyfunctional memory CD8 T cells specific for cytomegalovirus (CMV) or Epstein-Barr virus (EBV) also expressed TOX, either with or without TCF-1. A similar phenotype was observed among HIV-specific CD8 T cells from individuals who maintained exceptional immune control of viral replication. Collectively, these data demonstrate that TOX is expressed by most circulating effector memory CD8 T cell subsets and not exclusively linked to exhaustion.
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http://dx.doi.org/10.1126/sciimmunol.aba7918DOI Listing
July 2020

Jigsaw falling into place: A review and perspective of lymphoid tissue CD8+ T cells and control of HIV.

Mol Immunol 2020 08 8;124:42-50. Epub 2020 Jun 8.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 19104, USA. Electronic address:

CD8+ T cells are crucial for immunity against viral infections, including HIV. Several characteristics of CD8+ T cells, such as polyfunctionality and cytotoxicity, have been correlated with effective control of HIV. However, most of these correlates have been established in the peripheral blood. Meanwhile, HIV primarily replicates in lymphoid tissues. Therefore, it is unclear which aspects of CD8+ T cell biology are shared and which are different between blood and lymphoid tissues in the context of HIV infection. In this review, we will recapitulate the latest advancements of our knowledge on lymphoid tissue CD8+ T cells during HIV infection and discuss the insights these advancements might provide for the development of a HIV cure.
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http://dx.doi.org/10.1016/j.molimm.2020.05.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7279761PMC
August 2020

Erythrocytes Reveal Complement Activation in Patients with COVID-19.

medRxiv 2020 May 22. Epub 2020 May 22.

COVID-19, the disease caused by the SARS-CoV-2 virus, can progress to multi-organ failure characterized by respiratory insufficiency, arrhythmias, thromboembolic complications and shock. The mortality of patients hospitalized with COVID-19 is unacceptably high and new strategies are urgently needed to rapidly identify and treat patients at risk for organ failure. Clinical epidemiologic studies demonstrate that vulnerability to organ failure is greatest after viral clearance from the upper airway, which suggests that dysregulation of the host immune response is a critical mediator of clinical deterioration and death. Autopsy and pre-clinical evidence implicate aberrant complement activation in endothelial injury and organ failure. A potential therapeutic strategy warranting investigation is to inhibit complement, with case reports of successful treatment of COVID-19 with inhibitors of complement. However, this approach requires careful balance between the host protective and potential injurious effects of complement activation, and biomarkers to identify the optimal timing and candidates for therapy are lacking. Here we report the presence of complement activation products on circulating erythrocytes from hospitalized COVID-19 patients using flow cytometry. These findings suggest that novel erythrocyte-based diagnostics provide a method to identify patients with dysregulated complement activation.
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http://dx.doi.org/10.1101/2020.05.20.20104398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7274235PMC
May 2020

Immunologic perturbations in severe COVID-19/SARS-CoV-2 infection.

bioRxiv 2020 May 18. Epub 2020 May 18.

Although critical illness has been associated with SARS-CoV-2-induced hyperinflammation, the immune correlates of severe COVID-19 remain unclear. Here, we comprehensively analyzed peripheral blood immune perturbations in 42 SARS-CoV-2 infected and recovered individuals. We identified broad changes in neutrophils, NK cells, and monocytes during severe COVID-19, suggesting excessive mobilization of innate lineages. We found marked activation within T and B cells, highly oligoclonal B cell populations, profound plasmablast expansion, and SARS-CoV-2-specific antibodies in many, but not all, severe COVID-19 cases. Despite this heterogeneity, we found selective clustering of severe COVID-19 cases through unbiased analysis of the aggregated immunological phenotypes. Our findings demonstrate broad immune perturbations spanning both innate and adaptive leukocytes that distinguish dysregulated host responses in severe SARS-CoV-2 infection and warrant therapeutic investigation.

One Sentence Summary: Broad immune perturbations in severe COVID-19.
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http://dx.doi.org/10.1101/2020.05.18.101717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263541PMC
May 2020

Deep immune profiling of COVID-19 patients reveals patient heterogeneity and distinct immunotypes with implications for therapeutic interventions.

bioRxiv 2020 May 23. Epub 2020 May 23.

Institute for Immunology, University of Pennsylvania Perelman School of Medicine.

COVID-19 has become a global pandemic. Immune dysregulation has been implicated, but immune responses remain poorly understood. We analyzed 71 COVID-19 patients compared to recovered and healthy subjects using high dimensional cytometry. Integrated analysis of ~200 immune and >30 clinical features revealed activation of T cell and B cell subsets, but only in some patients. A subgroup of patients had T cell activation characteristic of acute viral infection and plasmablast responses could reach >30% of circulating B cells. However, another subgroup had lymphocyte activation comparable to uninfected subjects. Stable versus dynamic immunological signatures were identified and linked to trajectories of disease severity change. These analyses identified three "immunotypes" associated with poor clinical trajectories versus improving health. These immunotypes may have implications for therapeutics and vaccines.
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http://dx.doi.org/10.1101/2020.05.20.106401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263500PMC
May 2020

Type I IFN response associated with mTOR activation in the TAFRO subtype of idiopathic multicentric Castleman disease.

JCI Insight 2020 05 7;5(9). Epub 2020 May 7.

Department of Medicine and.

The TAFRO clinical subtype of idiopathic multicentric Castleman disease (iMCD-TAFRO) is a rare hematologic illness involving episodic disease flares of thrombocytopenia, anasarca, fever, reticulin myelofibrosis, renal dysfunction, and organomegaly (TAFRO) and progressive multiple organ dysfunction. We previously showed that the mTOR signaling pathway is elevated in lymph nodes of iMCD-TAFRO patients and that an mTOR inhibitor is effective in a small cohort of patients. However, the upstream mechanisms, cell types, and mediators involved in disease pathogenesis remain unknown. Here, we developed a targeted approach to identify candidate cellular drivers and mechanisms in iMCD-TAFRO through cellular and transcriptomic studies. Using paired iMCD-TAFRO PBMC samples collected during flare and remission, we identified T cell activation and alterations in NK cell and monocyte subset frequencies during iMCD-TAFRO flare. These changes were associated with increased Type I IFN (IFN-I) response gene signatures across CD8+ T cells, NK cells, and monocytes. Finally, we found that IFN-β stimulation of monocytes and T cells from iMCD-TAFRO patient remission samples induced increased mTOR activation compared with healthy donors, and this was abrogated with either mTORC1 or JAK1/2 inhibition. The data presented here support a potentially novel role for IFN-I signaling as a driver of increased mTOR signaling in iMCD-TAFRO.
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http://dx.doi.org/10.1172/jci.insight.135031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253017PMC
May 2020

The Transcription Factor T-bet Resolves Memory B Cell Subsets with Distinct Tissue Distributions and Antibody Specificities in Mice and Humans.

Immunity 2020 05 29;52(5):842-855.e6. Epub 2020 Apr 29.

Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

B cell subsets expressing the transcription factor T-bet are associated with humoral immune responses and autoimmunity. Here, we examined the anatomic distribution, clonal relationships, and functional properties of T-bet and T-bet memory B cells (MBCs) in the context of the influenza-specific immune response. In mice, both T-bet and T-bet hemagglutinin (HA)-specific B cells arose in germinal centers, acquired memory B cell markers, and persisted indefinitely. Lineage tracing and IgH repertoire analyses revealed minimal interconversion between T-bet and T-bet MBCs, and parabionts showed differential tissue residency and recirculation properties. T-bet MBCs could be subdivided into recirculating T-bet MBCs and spleen-resident T-bet MBCs. Human MBCs displayed similar features. Conditional gene deletion studies revealed that T-bet expression in B cells was required for nearly all HA stalk-specific IgG2c antibodies and for durable neutralizing titers to influenza. Thus, T-bet expression distinguishes MBC subsets that have profoundly different homing, residency, and functional properties, and mediate distinct aspects of humoral immune memory.
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http://dx.doi.org/10.1016/j.immuni.2020.03.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7242168PMC
May 2020

High throughput pMHC-I tetramer library production using chaperone-mediated peptide exchange.

Nat Commun 2020 04 20;11(1):1909. Epub 2020 Apr 20.

Department of Chemistry and Biochemistry, University of California Santa Cruz, 1156 High St., Santa Cruz, CA, 95064, USA.

Peptide exchange technologies are essential for the generation of pMHC-multimer libraries used to probe diverse, polyclonal TCR repertoires in various settings. Here, using the molecular chaperone TAPBPR, we develop a robust method for the capture of stable, empty MHC-I molecules comprising murine H2 and human HLA alleles, which can be readily tetramerized and loaded with peptides of choice in a high-throughput manner. Alternatively, catalytic amounts of TAPBPR can be used to exchange placeholder peptides with high affinity peptides of interest. Using the same system, we describe high throughput assays to validate binding of multiple candidate peptides on empty MHC-I/TAPBPR complexes. Combined with tetramer-barcoding via a multi-modal cellular indexing technology, ECCITE-seq, our approach allows a combined analysis of TCR repertoires and other T cell transcription profiles together with their cognate antigen specificities in a single experiment. The new approach allows TCR/pMHC interactions to be interrogated easily at large scale.
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http://dx.doi.org/10.1038/s41467-020-15710-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7170893PMC
April 2020

Elite control of HIV is associated with distinct functional and transcriptional signatures in lymphoid tissue CD8 T cells.

Sci Transl Med 2019 12;11(523)

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

The functional properties of circulating CD8 T cells have been associated with immune control of HIV. However, viral replication occurs predominantly in secondary lymphoid tissues, such as lymph nodes (LNs). We used an integrated single-cell approach to characterize effective HIV-specific CD8 T cell responses in the LNs of elite controllers (ECs), defined as individuals who suppress viral replication in the absence of antiretroviral therapy (ART). Higher frequencies of total memory and follicle-homing HIV-specific CD8 T cells were detected in the LNs of ECs compared with the LNs of chronic progressors (CPs) who were not receiving ART. Moreover, HIV-specific CD8 T cells potently suppressed viral replication without demonstrable cytolytic activity in the LNs of ECs, which harbored substantially lower amounts of CD4 T cell-associated HIV DNA and RNA compared with the LNs of CPs. Single-cell RNA sequencing analyses further revealed a distinct transcriptional signature among HIV-specific CD8 T cells from the LNs of ECs, typified by the down-regulation of inhibitory receptors and cytolytic molecules and the up-regulation of multiple cytokines, predicted secreted factors, and components of the protein translation machinery. Collectively, these results provide a mechanistic framework to expedite the identification of novel antiviral factors, highlighting a potential role for the localized deployment of noncytolytic functions as a determinant of immune efficacy against HIV.
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http://dx.doi.org/10.1126/scitranslmed.aax4077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265335PMC
December 2019

Identifying and targeting pathogenic PI3K/AKT/mTOR signaling in IL-6-blockade-refractory idiopathic multicentric Castleman disease.

J Clin Invest 2019 08 13;129(10):4451-4463. Epub 2019 Aug 13.

Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

Background: Idiopathic multicentric Castleman disease (iMCD) is a hematologic illness involving cytokine-induced lymphoproliferation, systemic inflammation, cytopenias, and life-threatening multi-organ dysfunction. The molecular underpinnings of interleukin-6(IL-6)-blockade refractory patients remain unknown; no targeted therapies exist. In this study, we searched for therapeutic targets in IL-6-blockade refractory iMCD patients with the thrombocytopenia, anasarca, fever/elevated C-reactive protein, reticulin myelofibrosis, renal dysfunction, organomegaly (TAFRO) clinical subtype.

Methods: We analyzed tissues and blood samples from three IL-6-blockade refractory iMCD-TAFRO patients. Cytokine panels, quantitative serum proteomics, flow cytometry of PBMCs, and pathway analyses were employed to identify novel therapeutic targets. To confirm elevated mTOR signaling, a candidate therapeutic target from the above assays, immunohistochemistry was performed for phosphorylated S6, a read-out of mTOR activation, in three iMCD lymph node tissue samples and controls. Proteomic, immunophenotypic, and clinical response assessments were performed to quantify the effects of administration of the mTOR inhibitor, sirolimus.

Results: Studies of three IL-6-blockade refractory iMCD cases revealed increased CD8+ T cell activation, VEGF-A, and PI3K/Akt/mTOR pathway activity. Administration of sirolimus significantly attenuated CD8+ T cell activation and decreased VEGF-A levels. Sirolimus induced clinical benefit responses in all three patients with durable and ongoing remissions of 66, 19, and 19 months.

Conclusion: This precision medicine approach identifies PI3K/Akt/mTOR signaling as the first pharmacologically-targetable pathogenic process in IL-6-blockade refractory iMCD. Prospective evaluation of sirolimus in treatment-refractory iMCD is planned (NCT03933904).

Funding: Castleman's Awareness & Research Effort/Castleman Disease Collaborative Network, Penn Center for Precision Medicine, University Research Foundation, Intramural NIH funding, and National Heart Lung and Blood Institute.
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http://dx.doi.org/10.1172/JCI126091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6763254PMC
August 2019

Human CD4CD103 cutaneous resident memory T cells are found in the circulation of healthy individuals.

Sci Immunol 2019 07;4(37)

Department of Biosciences, University of Salzburg, Salzburg, Austria.

Tissue-resident memory T cells (T) persist locally in nonlymphoid tissues where they provide frontline defense against recurring insults. T at barrier surfaces express the markers CD103 and/or CD69, which function to retain them in epithelial tissues. In humans, neither the long-term migratory behavior of T nor their ability to reenter the circulation and potentially migrate to distant tissue sites has been investigated. Using tissue explant cultures, we found that CD4CD69CD103 T in human skin can down-regulate CD69 and exit the tissue. In addition, we identified a skin-tropic CD4CD69CD103 population in human lymph and blood that is transcriptionally, functionally, and clonally related to the CD4CD69CD103 T population in the skin. Using a skin xenograft model, we confirmed that a fraction of the human cutaneous CD4CD103 T population can reenter circulation and migrate to secondary human skin sites where they reassume a T phenotype. Thus, our data challenge current concepts regarding the strict tissue compartmentalization of CD4 T cell memory in humans.
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http://dx.doi.org/10.1126/sciimmunol.aav8995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7057121PMC
July 2019

T follicular helper cells in human efferent lymph retain lymphoid characteristics.

J Clin Invest 2019 07 2;129(8):3185-3200. Epub 2019 Jul 2.

Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

T follicular helper cells (Tfh), a subset of CD4+ T cells, provide requisite help to B cells in the germinal centers (GC) of lymphoid tissue. GC Tfh are identified by high expression of the chemokine receptor CXCR5 and the inhibitory molecule PD-1. Although more accessible, blood contains lower frequencies of CXCR5+ and PD-1+ cells that have been termed circulating Tfh (cTfh). However, it remains unclear whether GC Tfh exit lymphoid tissues and populate this cTfh pool. To examine exiting cells, we assessed the phenotype of Tfh present within the major conduit of efferent lymph from lymphoid tissues into blood, the human thoracic duct. Unlike what was found in blood, we consistently identified a CXCR5-bright PD-1-bright (CXCR5BrPD-1Br) Tfh population in thoracic duct lymph (TDL). These CXCR5BrPD-1Br TDL Tfh shared phenotypic and transcriptional similarities with GC Tfh. Moreover, components of the epigenetic profile of GC Tfh could be detected in CXCR5BrPD-1Br TDL Tfh and the transcriptional imprint of this epigenetic signature was enriched in an activated cTfh subset known to contain vaccine-responding cells. Together with data showing shared TCR sequences between the CXCR5BrPD-1Br TDL Tfh and cTfh, these studies identify a population in TDL as a circulatory intermediate connecting the biology of Tfh in blood to Tfh in lymphoid tissue.
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http://dx.doi.org/10.1172/JCI125628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6668682PMC
July 2019

Distinct phenotype and function of circulating Vδ1+ and Vδ2+ γδT-cells in acute and chronic hepatitis B.

PLoS Pathog 2019 04 18;15(4):e1007715. Epub 2019 Apr 18.

Department of Internal Medicine, University of Michigan, Ann Arbor MI, United States of America.

Hepatitis B virus (HBV) persists with global and virus-specific T-cell dysfunction, without T-cell based correlates of outcomes. To determine if γδT-cells are altered in HBV infection relative to clinical status, we examined the frequency, phenotype and function of peripheral blood Vδ1+ and Vδ2+γδT-cells by multi-parameter cytometry in a clinically diverse North American cohort of chronic hepatitis B (CHB), acute hepatitis B (AHB) and uninfected control subjects. We show that circulating γδT-cells were comprised predominantly of CD3hiCD4- Vδ2+γδT-cells with frequencies that were 2-3 fold higher among Asian than non-Asian Americans and inversely correlated with age, but without differences between CHB, AHB and control subjects. However, compared to control subjects, CHB was associated with increased TbethiEomesdim phenotype in Vδ2+γδT-cells whereas AHB was associated with increased TbethiEomesdim phenotype in Vδ1+γδT-cells, with significant correlations between Tbet/Eomes expression in γδT-cells with their expression of NK and T-cell activation and regulatory markers. As for effector functions, IFNγ/TNF responses to phosphoantigens or PMA/Ionomycin in Vδ2+γδT-cells were weaker in AHB but preserved in CHB, without significant differences for Vδ1+γδT-cells. Furthermore, early IFNγ/TNF responses in Vδ2+ γδT-cells to brief PMA/Ionomycin stimulation correlated inversely with serum ALT but not HBV DNA. Accordingly, IFNγ/TNF responses in Vδ2+γδT-cells were weaker in patients with CHB with hepatitis flare compared to those without hepatitis flares, and this functional deficit persisted beyond clinical resolution of CHB flare. We conclude that circulating γδT-cells show distinct activation and differentiatiation in acute and chronic HBV infection as part of lymphoid stress surveillance with potential role in clinical outcomes.
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http://dx.doi.org/10.1371/journal.ppat.1007715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6490945PMC
April 2019