Amy E. Baxter

University of Pennsylvania | UP · Department of Microbiology (Medicine)

Macrophages contribute to HIV-1 pathogenesis by forming a viral reservoir and mediating neurological disorders. Cell-free HIV-1 infection of macrophages is inefficient, in part due to low plasma membrane expression of viral entry receptors. We find that macrophages selectively capture and engulf HIV-1-infected CD4(+) T cells leading to efficient ma...

HIV cure efforts are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in patients receiving antiretroviral therapy. We combine fluorescent in situ RNA hybridization with detection of HIV protein and flow cytometry, enabling detection of 0.5-1 gag-p...

Immune profiling of COVID-19 patients Coronavirus disease 2019 (COVID-19) has affected millions of people globally, yet how the human immune system responds to and influences COVID-19 severity remains unclear. Mathew et al. present a comprehensive atlas of immune modulation associated with COVID-19. They performed high-dimensional flow cytometry of...

CD8+ T cell exhaustion (Tex) limits disease control during chronic viral infections and cancer. Here, we investigated the epigenetic factors mediating major chromatin-remodeling events in Tex-cell development. A protein-domain-focused in vivo CRISPR screen identified distinct functions for two versions of the SWI/SNF chromatin-remodeling complex in...

Simple, efficient and well-tolerated delivery of CRISPR genome editing systems into primary cells remains a major challenge. Here we describe an engineered Peptide-Assisted Genome Editing (PAGE) CRISPR–Cas system for rapid and robust editing of primary cells with minimal toxicity. The PAGE system requires only a 30-min incubation with a cell-penetr...

Identifying molecular mechanisms of exhausted CD8 T cells (Tex) is a key goal of improving immunotherapy of cancer and other diseases. However, high-throughput interrogation of in vivo Tex can be costly and inefficient. In vitro models of Tex are easily customizable and quickly generate high cellular yield, enabling CRISPR screening and other high-...

CD8+ T cell exhaustion (TEX) impairs the ability of T cells to clear chronic infection or cancer. While TEX are hypofunctional, some TEX retain effector gene signatures, a feature associated with killer lectin-like receptor (KLR) expression. Although KLR+ TEX (TKLR) may improve control of chronic antigen, the signaling molecules regulating this pop...

Identifying novel molecular mechanisms of exhausted CD8 T cells (Tex) is a key goal of improving immunotherapy of cancer and other diseases. However, high-throughput interrogation of in vivo Tex can be costly and inefficient. In vitro models of Tex are easily customizable and quickly generate high cellular yield, offering an opportunity to perform...

T cell exhaustion (TEX) impairs the ability of T cells to clear chronic infection or cancer. While exhausted T cells are hypofunctional, some exhausted T cells retain effector gene signatures, a feature that is associated with expression of KLRs (killer lectin-like receptors). Although KLR+ T cells may improve control of chronic antigen, the signal...

SARS-CoV-2 infection of vaccinated individuals is increasingly common but rarely results in severe disease, likely due to the enhanced potency and accelerated kinetics of memory immune responses. However, there have been few opportunities to rigorously study early recall responses during human viral infection. To better understand human immune memo...

Naïve CD8⁺ T cells can differentiate into effector (Teff), memory (Tmem) or exhausted (Tex) T cells. These developmental pathways are associated with distinct transcriptional and epigenetic changes that endow cells with different functional capacities and therefore therapeutic potential. The molecular circuitry underlying these developmental trajec...

Background Immune checkpoint blockade (ICB) partially reverses the dysfunctional state of antigen-specific T cell in chronic infections. However, its impact on the diverse subsets of CD4+ T cells in humans is largely unknown. Methods We examined immune checkpoint (IC) expression and function in HIV-specific CD4+ T cells of viremic individuals (≥50...

We examined antibody and memory B cell responses longitudinally for ∼9-10 months after primary 2-dose SARS-CoV-2 mRNA vaccination and 3 months after a 3rd dose. Antibody decay stabilized between 6 to 9 months and antibody quality continued to improve for at least 9 months after 2-dose vaccination. Spike- and RBD-specific memory B cells remained dur...

Naive CD8 T cells can differentiate into effector (TEFF), memory (TMEM), or exhausted (TEX) CD8 T cells. These developmental pathways are associated with distinct transcriptional and epigenetic changes that endow cells with different functional capacities and therefore therapeutic potential. The molecular circuitry underlying these developmental tr...

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of infl...

The clinical benefit of T cell immunotherapies remains limited by incomplete understanding of T cell differentiation and dysfunction. We generated an epigenetic and transcriptional atlas of T cell differentiation from healthy humans that included exhausted CD8 T cells and applied this resource in three ways. First, we identified modules of gene exp...

Despite a clear role in protective immunity, the durability and quality of antibody and memory B cell responses induced by mRNA vaccination, particularly by a 3 rd dose of vaccine, remains unclear. Here, we examined antibody and memory B cell responses in a cohort of individuals sampled longitudinally for ∼9-10 months after the primary 2-dose mRNA...

Multi-system Inflammatory Syndrome in Children (MIS-C) is a major complication of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection in pediatric patients. Weeks after an often mild or asymptomatic initial infection with SARS-CoV-2 children may present with a severe shock-like picture and marked inflammation. Children with MIS-C...

Immune memory after vaccination Vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has proven highly effective at preventing severe COVID-19. However, the evolution of viral variants, and waning antibody levels over time, raise questions regarding the longevity of vaccine-induced immune protection. Goel et al . examine...

Background: Antigen-specific T cell impairment is observed in chronic infections. CD4+ T cells are diverse in phenotype and function; how their different lineages are impacted by inhibitory immune checkpoints (IC) is unknown. Methods: We examined IC expression and function in HIV-specific CD4+ T cells of viremic individuals prior to ART initiation...

SARS-CoV-2 mRNA vaccines have shown remarkable efficacy, especially in preventing severe illness and hospitalization. However, the emergence of several variants of concern and reports of declining antibody levels have raised uncertainty about the durability of immune memory following vaccination. In this study, we longitudinally profiled both antib...

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of the respiratory tract, results in highly variable outcomes ranging from minimal illness to death, but the reasons for this are not well understood. We investigated the respiratory tract bacterial microbiome and small commensal DNA viruses in hospitalized C...

SARS-CoV-2 mRNA vaccines have shown remarkable clinical efficacy, but questions remain about the nature and kinetics of T cell priming. We performed longitudinal antigen-specific T cell analyses on healthy SARS-CoV-2-naive and recovered individuals prior to and following mRNA prime and boost vaccination. Vaccination induced rapid antigen-specific C...

Although understanding the diversity of HIV-1 reservoirs is key to achieving a cure, their study at the single-cell level in primary samples remains challenging. We combine flow cytometric multiplexed fluorescent in situ RNA hybridization for different viral genes with HIV-1 p24 protein detection, cell phenotyping, and downstream near-full-length s...

Patients with cancer have high mortality from coronavirus disease 2019 (COVID-19), and the immune parameters that dictate clinical outcomes remain unknown. In a cohort of 100 patients with cancer who were hospitalized for COVID-19, patients with hematologic cancer had higher mortality relative to patients with solid cancer. In two additional cohort...

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of infl...

CD8 T cells are critical for clearing infections and immune surveillance of cancers. However, in the setting of chronic viral infections and tumor growth, CD8 T cells become exhausted. T cell exhaustion (TEX) is characterized by a progressive increase of inhibitory receptor expression and concomitant diminishment of effector function. This diminish...

The SARS-CoV-2 mRNA vaccines have shown remarkable clinical efficacy, but questions remain about the nature and kinetics of T cell priming. We performed longitudinal antigen-specific T cell analyses in healthy individuals following mRNA vaccination. Vaccination induced rapid near-maximal antigen-specific CD4 ⁺ T cell responses in all subjects after...

Multi-system Inflammatory Syndrome in Children (MIS-C) is a major complication of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic in pediatric patients. Weeks after an often mild or asymptomatic initial infection with SARS-CoV-2 children may present with a severe shock-like picture and marked inflammation. Children with MI...

Novel mRNA vaccines for SARS-CoV-2 have been authorized for emergency use. Despite their efficacy in clinical trials, data on mRNA vaccine-induced immune responses are mostly limited to serological analyses. Here, we interrogated antibody and antigen-specific memory B cells over time in 33 SARS-CoV-2 naïve and 11 SARS-CoV-2 recovered subjects. SARS...

Rationale: Viral infection of the respiratory tract can be associated with propagating effects on the airway microbiome, and microbiome dysbiosis may influence viral disease. Objective: To define the respiratory tract microbiome in COVID-19 and relationship disease severity, systemic immunologic features, and outcomes. Methods and Measurements: We...

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 m...

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...

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 COVID-19 pandemic. Here, we quantified levels of SARS-CoV-2-reactive a...

Improving effector activity of antigen-specific T cells is a major goal in cancer immunotherapy. Despite the identification of several effector T cell (TEFF)-driving transcription factors (TFs), the transcriptional coordination of TEFF biology remains poorly understood. We developed an in vivo T cell CRISPR screening platform and identified a key m...

Cancer patients have increased morbidity and mortality from Coronavirus Disease 2019 (COVID-19), but the underlying immune mechanisms are unknown. In a cohort of 100 cancer patients hospitalized for COVID-19 at the University of Pennsylvania Health System, we found that patients with hematologic cancers had a significantly higher mortality relative...

SARS-CoV-2 antibody responses in children remain poorly characterized. Here, we show that pediatric patients with multisystem inflammatory syndrome in children (MIS-C) possess higher SARS-CoV-2 spike IgG titers compared to those with severe coronavirus disease 2019 (COVID-19), likely reflecting a longer time since onset of infection in MIS-C patien...

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 COVID-19 pandemic. Here, we quantified levels of SARS-CoV-2-reactive a...

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...

SARS-CoV-2 antibody responses in children remain poorly characterized. Here, we show that pediatric patients with multisystem inflammatory syndrome in children (MIS-C) possess higher SARS-CoV-2 spike IgG titers compared to those with severe coronavirus disease 2019 (COVID-19), likely reflecting a longer time since onset of infection in MIS-C patien...

Limited data are available for pregnant women affected by SARS-CoV-2. Serological tests are critically important for determining SARS-CoV-2 exposures within both individuals and populations. We validated a SARS-CoV-2 spike receptor binding domain serological test using 834 pre-pandemic samples and 31 samples from COVID-19 recovered donors. We then...

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, i...

Limited data are available for pregnant women affected by SARS-CoV-2. Serological tests are critically important to determine exposure and immunity to SARS-CoV-2 within both individuals and populations. We completed SARS-CoV-2 serological testing of 1,293 parturient women at two centers in Philadelphia from April 4 to June 3, 2020. We tested 834 pr...

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, b...

Improving effector activity of antigen specific T cells is a major goal in cancer immunotherapy. Despite the identification of several effector T cell (TEFF)-driving transcription factors (TF), the transcriptional coordination of TEFF biology remains poorly understood. We developed an in vivo T cell CRISPR screening platform and identified a novel...

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 seve...

SARS-CoV-2 has led to a pandemic of respiratory and multisystem disease, named COVID-19.1 Limited data are available for pregnant women affected by COVID-19.2 Serological tests, particularly those that provide quantitative information, are critically important to determine exposure and immunity to SARS-CoV-2 within both individuals and populations....

Background Untreated HIV infection leads to alterations in HIV-specific CD4⁺ T cells including increased expression of co-inhibitory receptors (IRs) and skewing toward a T follicular helper cell (Tfh) signature. However, which changes are maintained after suppression of viral replication with antiretroviral therapy (ART) is poorly known. Methods W...

Combination antiretroviral therapy (ART) is highly effective in controlling human immunodeficiency virus (HIV)-1 but requires lifelong medication due to the existence of a latent viral reservoir. Potent broadly neutralizing antibodies (bNAbs) represent a potential alternative or adjuvant to ART. In addition to suppressing viremia, bNAbs may have T...

Chronic viral infections disrupt B cell responses leading to impaired affinity maturation and delayed control of viremia. Previous studies have identified early pre-germinal center (GC) B cell attrition but the impact of chronic infections on B cell fate decisions in the GC remains poorly understood. To address this question, we used single-cell tr...

TCF-1 is a key transcription factor in progenitor exhausted CD8 T cells (Tex). Moreover, this Tex cell subset mediates responses to PD-1 checkpoint pathway blockade. However, the role of the transcription factor TCF-1 in early fate decisions and initial generation of Tex cells is unclear. Single-cell RNA sequencing (scRNA-seq) and lineage tracing i...

Dysfunction of virus-specific CD4⁺ T cells in chronic human infections is poorly understood. We performed genome-wide transcriptional analyses and functional assays of CD4⁺ T cells specific for human immunodeficiency virus (HIV) from HIV-infected people before and after initiation of antiretroviral therapy (ART). A follicular helper T cell (TFH cel...

The phenotypic characterization of the cells in which HIV persists during antiretroviral therapy (ART) remains technically challenging. We developed a simple flow cytometry-based assay to quantify and characterize infected cells producing HIV proteins during untreated and treated HIV infection. By combining two antibodies targeting the HIV capsid i...

MFI of p24 antibodies following stimulation. Comparison of the MFI of the two p24 antibodies (p24 28B7-APC and p24 KC57-PE) in the presence or absence of stimulation with PMA/ionomycin in samples from 6 untreated individuals. The MFI of p24 antibodies was measured within the p24+ gate (p24 KC57+/p24 28B7+). (TIF)

Comparison of two permeabilization buffers for the detection of p24-producing cells by HIV-Flow. Dot plots showing the detection of p24+ cells in 2 samples, using 2 experimental conditions. Purified CD4 T cells from a viremic individual were rested for 18 hours, while purified CD4 T cells from an ART-suppressed individual were stimulated with PMA/i...

Markers showing significant changes of expression following stimulation. (A) Representative dot plots showing the levels of expression of CXCR3/CCR4/CCR6 after 24h of resting or after 24h of stimulation with PMA/ionomycin + BFA in one representative ART-suppressed individual. (B) As in A) for CXCR5 and CD25. (C) As in A) for CD3 and CD4. Of note, t...

Gating strategy used in HIV-Flow. Example of the gating strategy used for a representative sample following PMA/ionomycin stimulation of CD4+ T cells obtained from an ART-suppressed individual. (TIF)

Frequencies of infected cells measured by different assays in samples from ART-suppressed individuals. (DOCX)

Characterization of p24 KC57 and p24 28B7 antibodies. (A) Representative dot plots obtained from a binding competition experiment. Staining with either KC57 or 28B7 in a first step (1) does not prevent subsequent staining with the other antibody (2) (middle and right panels). Co-staining with both antibodies yielded a similar frequency of p24+ cell...

Single positive cells contain low HIV DNA levels. (A) Representative dot plot showing the gating strategy used to sort four populations of unstimulated cells (KC57+/28B7+, KC57+, 28B7+ and KC57-/28B7- cells) obtained from one untreated individual (VIR21). Total HIV DNA was quantified by ultrasensitive PCR in each sorted subset (right). (B) Levels o...

HIV DNA detection by PCR in p24+ single sorted cells. p24- and p24+ CD4 T cells from three ART-suppressed individuals were single sorted by flow cytometry and subjected to a duplex ultrasensitive PCR for the CD3 gene and the HIV genome (LTR/gag). Grey and dark circles represent successful detection of the CD3 gene and the HIV genome, respectively....

Boolean analysis. (A) Frequencies of p24+ cells in all cells and in cell subsets expressing 0, 1, 2, 3 or 4 markers in samples from 8 viremic individuals (same as in Figs 4 and 5). Analyses were performed on cells expressing CD25/CD95/HLA-DR/Ki-67 (top panel) and PD-1/TIGIT/LAG-3/Tim-3 (middle panel). (B) Frequencies of p24+ cells in all cells and...

p24-producing cells from ART-suppressed individuals are highly enriched in CD45RA-α4β1+TIGIT+ cells. Frequencies of p24+ cells in all cells and in each gated cellular subset in samples from 8 ART-suppressed individuals. Each sample is represented by a unique color-coded symbol. Undetectable measurements are represented as open symbols. Mean folds-e...

Median fold differences in the frequencies of p24+ cells and all cells expressing a given cellular marker. (DOCX)

Frequencies of p24+ cells in different subsets. (A) Frequencies of p24+ cells in all cells and in each gated cellular subset in samples from 8 viremic individuals (same as in Figs 4 and 5). (B) Frequencies of p24+ cells in all cells and in each gated cellular subset in samples from 12 virally suppressed individuals (same as in Fig 6). Each sample i...

Contribution of different subsets to the pool of p24+ cells. (A) Pie charts comparing the relative contributions of different subsets to the total pool of CD4 T cells (all cells, left) and to the pool of p24+ cells (right) in samples from viremic individuals. Contributions of memory subsets and effector subsets are represented. (B) Pie charts compa...