Longitudinal Multi-omic Immune Profiling Reveals Age-Related Immune Cell Dynamics in Healthy Adults.

The generation and maintenance of protective immunity is a dynamic interplay between host and environment that is impacted by age. Understanding fundamental changes in the healthy immune system that occur over a lifespan is critical in developing interventions for age-related susceptibility to infections and diseases. Here, we use multi-omic profiling (scRNA-seq, proteomics, flow cytometry) to examined human peripheral immunity in over 300 healthy adults, with 96 young and older adults followed over two years with yearly vaccination. The resulting resource includes scRNA-seq datasets of >16 million PBMCs, interrogating 71 immune cell subsets from our new Immune Health Atlas. This study allows unique insights into the composition and transcriptional state of immune cells at homeostasis, with vaccine perturbation, and across age. We find that T cells specifically accumulate age-related transcriptional changes more than other immune cells, independent from inflammation and chronic perturbation. Moreover, impaired memory B cell responses to vaccination are linked to a Th2-like state shift in older adults' memory CD4 T cells, revealing possible mechanisms of immune dysregulation during healthy human aging. This extensive resource is provided with a suite of exploration tools at https://apps.allenimmunology.org/aifi/insights/dynamics-imm-health-age/ to enhance data accessibility and further the understanding of immune health across age.

LAG-3 and PD-1 synergize on CD8+ T cells to drive T cell exhaustion and hinder autocrine IFN-γ-dependent anti-tumor immunity.

Overcoming immune-mediated resistance to PD-1 blockade remains a major clinical challenge. Enhanced efficacy has been demonstrated in melanoma patients with combined nivolumab (anti-PD-1) and relatlimab (anti-LAG-3) treatment, the first in its class to be FDA approved. However, how these two inhibitory receptors synergize to hinder anti-tumor immunity remains unknown. Here, we show that CD8+ T cells deficient in both PD-1 and LAG-3, in contrast to CD8+ T cells lacking either receptor, mediate enhanced tumor clearance and long-term survival in mouse models of melanoma. PD-1- and LAG-3-deficient CD8+ T cells were transcriptionally distinct, with broad TCR clonality and enrichment of effector-like and interferon-responsive genes, resulting in enhanced IFN-γ release indicative of functionality. LAG-3 and PD-1 combined to drive T cell exhaustion, playing a dominant role in modulating TOX expression. Mechanistically, autocrine, cell-intrinsic IFN-γ signaling was required for PD-1- and LAG-3-deficient CD8+ T cells to enhance anti-tumor immunity, providing insight into how combinatorial targeting of LAG-3 and PD-1 enhances efficacy.

LAG-3 sustains TOX expression and regulates the CD94/NKG2-Qa-1b axis to govern exhausted CD8 T cell NK receptor expression and cytotoxicity.

Exhausted CD8 T (Tex) cells in chronic viral infection and cancer have sustained co-expression of inhibitory receptors (IRs). Tex cells can be reinvigorated by blocking IRs, such as PD-1, but synergistic reinvigoration and enhanced disease control can be achieved by co-targeting multiple IRs including PD-1 and LAG-3. To dissect the molecular changes intrinsic when these IR pathways are disrupted, we investigated the impact of loss of PD-1 and/or LAG-3 on Tex cells during chronic infection. These analyses revealed distinct roles of PD-1 and LAG-3 in regulating Tex cell proliferation and effector functions, respectively. Moreover, these studies identified an essential role for LAG-3 in sustaining TOX and Tex cell durability as well as a LAG-3-dependent circuit that generated a CD94/NKG2+ subset of Tex cells with enhanced cytotoxicity mediated by recognition of the stress ligand Qa-1b, with similar observations in humans. These analyses disentangle the non-redundant mechanisms of PD-1 and LAG-3 and their synergy in regulating Tex cells.

The future of immunology: a Lofoten perspective.

This Future Challenges article summarizes views on future directions in immunological research presented at round-table discussions at the 4th Immunology workshop in the Lofoten Islands in Norway, held in August 2023, and subsequent responses to surveys sent to meeting participants. It also summarizes some of the conversations around the responsibility of scientists to communicate with the non-science community, and the approaches that we may use to meet this obligation.

Immunologic Predictors of Vaccine Responsiveness in Patients With Lymphoma and Chronic Lymphocytic Leukemia.

Patients with B-cell lymphomas have altered cellular components of vaccine responses due to malignancy and therapy, and the optimal timing of vaccination relative to therapy remains unknown. Severe acute respiratory syndrome coronavirus 2 vaccines created an opportunity for new insights in vaccine timing because patients were challenged with a novel antigen across multiple phases of treatment. We studied serologic messenger RNA vaccine response in retrospective and prospective cohorts with lymphoma and chronic lymphocytic leukemia, paired with clinical and research immune parameters. Reduced serologic response was observed more frequently during active treatment, but nonresponse was also common within observation and posttreatment groups. Total immunoglobulin A and immunoglobulin M correlated with successful vaccine response. In individuals treated with anti-CD19-directed chimeric antigen receptor-modified T cells, nonresponse was associated with reduced B and T follicular helper cells. Predictors of vaccine response varied by disease and therapeutic group, and therefore further studies of immune health during and after cancer therapies are needed to individualize vaccine timing.

Dysregulated STAT3 signaling and T cell immunometabolic dysfunction define a targetable, high mortality subphenotype of critically ill children.

Sepsis is the leading cause of death of hospitalized children worldwide. Despite the established link between immune dysregulation and mortality in pediatric sepsis, it remains unclear which host immune factors contribute causally to adverse sepsis outcomes. Identifying modifiable pathobiology is an essential first step to successful translation of biologic insights into precision therapeutics. We designed a prospective, longitudinal cohort study of 88 critically ill pediatric patients with multiple organ dysfunction syndrome (MODS), including patients with and without sepsis, to define subphenotypes associated with targetable mechanisms of immune dysregulation. We first assessed plasma proteomic profiles and identified shared features of immune dysregulation in MODS patients with and without sepsis. We then employed consensus clustering to define three subphenotypes based on protein expression at disease onset and identified a strong association between subphenotype and clinical outcome. We next identified differences in immune cell frequency and activation state by MODS subphenotype and determined the association between hyperinflammatory pathway activation and cellular immunophenotype. Using single cell transcriptomics, we demonstrated STAT3 hyperactivation in lymphocytes from the sickest MODS subgroup and then identified an association between STAT3 hyperactivation and T cell immunometabolic dysregulation. Finally, we compared proteomics findings between patients with MODS and patients with inborn errors of immunity that amplify cytokine signaling pathways to further assess the impact of STAT3 hyperactivation in the most severe patients with MODS. Overall, these results identify a potentially pathologic and targetable role for STAT3 hyperactivation in a subset of pediatric patients with MODS who have high severity of illness and poor prognosis.

Combined JAK inhibition and PD-1 immunotherapy for non-small cell lung cancer patients.

Persistent inflammation driven by cytokines such as type-one interferon (IFN-I) can cause immunosuppression. We show that administration of the Janus kinase 1 (JAK1) inhibitor itacitinib after anti-PD-1 (programmed cell death protein 1) immunotherapy improves immune function and antitumor responses in mice and results in high response rates (67%) in a phase 2 clinical trial for metastatic non-small cell lung cancer. Patients who failed to respond to initial anti-PD-1 immunotherapy but responded after addition of itacitinib had multiple features of poor immune function to anti-PD-1 alone that improved after JAK inhibition. Itacitinib promoted CD8 T cell plasticity and therapeutic responses of exhausted and effector memory-like T cell clonotypes. Patients with persistent inflammation refractory to itacitinib showed progressive CD8 T cell terminal differentiation and progressive disease. Thus, JAK inhibition may improve the efficacy of anti-PD-1 immunotherapy by pivoting T cell differentiation dynamics.

Automated Cytometric Gating with Human-Level Performance Using Bivariate Segmentation.

Recent advances in cytometry technology have enabled high-throughput data collection with multiple single-cell protein expression measurements. The significant biological and technical variance between samples in cytometry has long posed a formidable challenge during the gating process, especially for the initial gates which deal with unpredictable events, such as debris and technical artifacts. Even with the same experimental machine and protocol, the target population, as well as the cell population that needs to be excluded, may vary across different measurements. To address this challenge and mitigate the labor-intensive manual gating process, we propose a deep learning framework UNITO to rigorously identify the hierarchical cytometric subpopulations. The UNITO framework transformed a cell-level classification task into an image-based semantic segmentation problem. For reproducibility purposes, the framework was applied to three independent cohorts and successfully detected initial gates that were required to identify single cellular events as well as subsequent cell gates. We validated the UNITO framework by comparing its results with previous automated methods and the consensus of at least four experienced immunologists. UNITO outperformed existing automated methods and differed from human consensus by no more than each individual human. Most critically, UNITO framework functions as a fully automated pipeline after training and does not require human hints or prior knowledge. Unlike existing multi-channel classification or clustering pipelines, UNITO can reproduce a similar contour compared to manual gating for each intermediate gating to achieve better interpretability and provide post hoc visual inspection. Beyond acting as a pioneering framework that uses image segmentation to do auto-gating, UNITO gives a fast and interpretable way to assign the cell subtype membership, and the speed of UNITO will not be impacted by the number of cells from each sample. The pre-gating and gating inference takes approximately 2 minutes for each sample using our pre-defined 9 gates system, and it can also adapt to any sequential prediction with different configurations.

Nivolumab for mismatch-repair-deficient or hypermutated gynecologic cancers: a phase 2 trial with biomarker analyses.

Programmed death-1 (PD-1) inhibitors are approved for therapy of gynecologic cancers with DNA mismatch repair deficiency (dMMR), although predictors of response remain elusive. We conducted a single-arm phase 2 study of nivolumab in 35 patients with dMMR uterine or ovarian cancers. Co-primary endpoints included objective response rate (ORR) and progression-free survival at 24 weeks (PFS24). Secondary endpoints included overall survival (OS), disease control rate (DCR), duration of response (DOR) and safety. Exploratory endpoints included biomarkers and molecular correlates of response. The ORR was 58.8% (97.5% confidence interval (CI): 40.7-100%), and the PFS24 rate was 64.7% (97.5% one-sided CI: 46.5-100%), meeting the pre-specified endpoints. The DCR was 73.5% (95% CI: 55.6-87.1%). At the median follow-up of 42.1 months (range, 8.9-59.8 months), median OS was not reached. One-year OS rate was 79% (95% CI: 60.9-89.4%). Thirty-two patients (91%) had a treatment-related adverse event (TRAE), including arthralgia (n = 10, 29%), fatigue (n = 10, 29%), pain (n = 10, 29%) and pruritis (n = 10, 29%); most were grade 1 or grade 2. Ten patients (29%) reported a grade 3 or grade 4 TRAE; no grade 5 events occurred. Exploratory analyses show that the presence of dysfunctional (CD8+PD-1+) or terminally dysfunctional (CD8+PD-1+TOX+) T cells and their interaction with programmed death ligand-1 (PD-L1)+ cells were independently associated with PFS24. PFS24 was associated with presence of MEGF8 or SETD1B somatic mutations. This trial met its co-primary endpoints (ORR and PFS24) early, and our findings highlight several genetic and tumor microenvironment parameters associated with response to PD-1 blockade in dMMR cancers, generating rationale for their validation in larger cohorts.ClinicalTrials.gov identifier: NCT03241745 .

Immunological imprinting shapes the specificity of human antibody responses against SARS-CoV-2 variants.

The spike glycoprotein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to accumulate substitutions, leading to breakthrough infections of vaccinated individuals. It remains unclear if exposures to antigenically distant SARS-CoV-2 variants can overcome memory B cell biases established by initial SARS-CoV-2 encounters. We determined the specificity and functionality of antibody and B cell responses following exposure to BA.5 and XBB variants in individuals who received ancestral SARS-CoV-2 mRNA vaccines. BA.5 exposures elicited antibody responses that targeted epitopes conserved between the BA.5 and ancestral spike. XBB exposures also elicited antibody responses that primarily targeted epitopes conserved between the XBB.1.5 and ancestral spike. However, unlike BA.5, a single XBB exposure elicited low frequencies of XBB.1.5-specific antibodies and B cells in some individuals. Pre-existing cross-reactive B cells and antibodies were correlated with stronger overall responses to XBB but weaker XBB-specific responses, suggesting that baseline immunity influences the activation of variant-specific SARS-CoV-2 responses.

Neonatal CD8+ T Cells Resist Exhaustion during Chronic Infection.

Chronic viral infections, such as HIV and hepatitis C virus, represent a major public health problem. Although it is well understood that neonates and adults respond differently to chronic viral infections, the underlying mechanisms remain unknown. In this study, we transferred neonatal and adult CD8+ T cells into a mouse model of chronic infection (lymphocytic choriomeningitis virus clone 13) and dissected out the key cell-intrinsic differences that alter their ability to protect the host. Interestingly, we found that neonatal CD8+ T cells preferentially became effector cells early in chronic infection compared with adult CD8+ T cells and expressed higher levels of genes associated with cell migration and effector cell differentiation. During the chronic phase of infection, the neonatal cells retained more immune functionality and expressed lower levels of surface markers and genes related to exhaustion. Because the neonatal cells protect from viral replication early in chronic infection, the altered differentiation trajectories of neonatal and adult CD8+ T cells is functionally significant. Together, our work demonstrates how cell-intrinsic differences between neonatal and adult CD8+ T cells influence key cell fate decisions during chronic infection.

Repeated peripheral infusions of anti-EGFRvIII CAR T cells in combination with pembrolizumab show no efficacy in glioblastoma: a phase 1 trial.

We previously showed that chimeric antigen receptor (CAR) T-cell therapy targeting epidermal growth factor receptor variant III (EGFRvIII) produces upregulation of programmed death-ligand 1 (PD-L1) in the tumor microenvironment (TME). Here we conducted a phase 1 trial (NCT03726515) of CAR T-EGFRvIII cells administered concomitantly with the anti-PD1 (aPD1) monoclonal antibody pembrolizumab in patients with newly diagnosed, EGFRvIII+ glioblastoma (GBM) (n = 7). The primary outcome was safety, and no dose-limiting toxicity was observed. Secondary outcomes included median progression-free survival (5.2 months; 90% confidence interval (CI), 2.9-6.0 months) and median overall survival (11.8 months; 90% CI, 9.2-14.2 months). In exploratory analyses, comparison of the TME in tumors harvested before versus after CAR + aPD1 administration demonstrated substantial evolution of the infiltrating myeloid and T cells, with more exhausted, regulatory, and interferon (IFN)-stimulated T cells at relapse. Our study suggests that the combination of CAR T cells and PD-1 inhibition in GBM is safe and biologically active but, given the lack of efficacy, also indicates a need to consider alternative strategies.

Immunological imprinting shapes the specificity of human antibody responses against SARS-CoV-2 variants.

The spike glycoprotein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to accumulate substitutions, leading to breakthrough infections of vaccinated individuals and prompting the development of updated booster vaccines. Here, we determined the specificity and functionality of antibody and B cell responses following exposure to BA.5 and XBB variants in individuals who received ancestral SARS-CoV-2 mRNA vaccines. BA.5 exposures elicited antibody responses that primarily targeted epitopes conserved between the BA.5 and ancestral spike, with poor reactivity to the XBB.1.5 variant. XBB exposures also elicited antibody responses that targeted epitopes conserved between the XBB.1.5 and ancestral spike. However, unlike BA.5, a single XBB exposure elicited low levels of XBB.1.5-specific antibodies and B cells in some individuals. Pre-existing cross-reactive B cells and antibodies were correlated with stronger overall responses to XBB but weaker XBB-specific responses, suggesting that baseline immunity influences the activation of variant-specific SARS-CoV-2 responses.

FDG PET/CT Imaging 1 Week after a Single Dose of Pembrolizumab Predicts Treatment Response in Patients with Advanced Melanoma.

PURPOSE: Immunologic response to anti-programmed cell death protein 1 (PD-1) therapy can occur rapidly with T-cell responses detectable in as little as one week. Given that activated immune cells are FDG avid, we hypothesized that an early FDG PET/CT obtained approximately 1 week after starting pembrolizumab could be used to visualize a metabolic flare (MF), with increased tumor FDG activity due to infiltration by activated immune cells, or a metabolic response (MR), due to tumor cell death, that would predict response. PATIENTS AND METHODS: Nineteen patients with advanced melanoma scheduled to receive pembrolizumab were prospectively enrolled. FDG PET/CT imaging was performed at baseline and approximately 1 week after starting treatment. FDG PET/CT scans were evaluated for changes in maximum standardized uptake value (SUVmax) and thresholds were identified by ROC analysis; MF was defined as >70% increase in tumor SUVmax, and MR as >30% decrease in tumor SUVmax. RESULTS: An MF or MR was identified in 6 of 11 (55%) responders and 0 of 8 (0%) nonresponders, with an objective response rate (ORR) of 100% in the MF-MR group and an ORR of 38% in the stable metabolism (SM) group. An MF or MR was associated with T-cell reinvigoration in the peripheral blood and immune infiltration in the tumor. Overall survival at 3 years was 83% in the MF-MR group and 62% in the SM group. Median progression-free survival (PFS) was >38 months (median not reached) in the MF-MR group and 2.8 months (95% confidence interval, 0.3-5.2) in the SM group (P = 0.017). CONCLUSIONS: Early FDG PET/CT can identify metabolic changes in melanoma metastases that are potentially predictive of response to pembrolizumab and significantly correlated with PFS.

Efficient engineering of human and mouse primary cells using peptide-assisted genome editing.

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-penetrating Cas9 or Cas12a and a cell-penetrating endosomal escape peptide to achieve robust single and multiplex genome editing. Unlike electroporation-based methods, PAGE gene editing has low cellular toxicity and shows no significant transcriptional perturbation. We demonstrate rapid and efficient editing of primary cells, including human and mouse T cells, as well as human hematopoietic progenitor cells, with editing efficiencies upwards of 98%. PAGE provides a broadly generalizable platform for next-generation genome engineering in primary cells.

SARS-CoV-2 vaccination enhances the effector qualities of spike-specific T cells induced by COVID-19.

T cells are critical for immune protection against severe COVID-19, but it has remained unclear whether repeated exposure to SARS-CoV-2 antigens delivered in the context of vaccination fuels T cell exhaustion or reshapes T cell functionality. Here, we sampled convalescent donors with a history of mild or severe COVID-19 before and after SARS-CoV-2 vaccination to profile the functional spectrum of hybrid T cell immunity. Using combined single-cell technologies and high-dimensional flow cytometry, we found that the frequencies and functional capabilities of spike-specific CD4+ and CD8+ T cells in previously infected individuals were enhanced by vaccination, despite concomitant increases in the expression of inhibitory receptors such as PD-1 and TIM3. In contrast, CD4+ and CD8+ T cells targeting non-spike proteins remained functionally static and waned over time, and only minimal effects were observed in healthy vaccinated donors experiencing breakthrough infections with SARS-CoV-2. Moreover, hybrid immunity was characterized by elevated expression of IFN-γ, which was linked with clonotype specificity in the CD8+ T cell lineage. Collectively, these findings identify a molecular hallmark of hybrid immunity and suggest that vaccination after infection is associated with cumulative immunological benefits over time, potentially conferring enhanced protection against subsequent episodes of COVID-19.

Stat5 opposes the transcription factor Tox and rewires exhausted CD8+ T cells toward durable effector-like states during chronic antigen exposure.

Rewiring exhausted CD8+ T (Tex) cells toward functional states remains a therapeutic challenge. Tex cells are epigenetically programmed by the transcription factor Tox. However, epigenetic remodeling occurs as Tex cells transition from progenitor (Texprog) to intermediate (Texint) and terminal (Texterm) subsets, suggesting development flexibility. We examined epigenetic transitions between Tex cell subsets and revealed a reciprocally antagonistic circuit between Stat5a and Tox. Stat5 directed Texint cell formation and re-instigated partial effector biology during this Texprog-to-Texint cell transition. Constitutive Stat5a activity antagonized Tox and rewired CD8+ T cells from exhaustion to a durable effector and/or natural killer (NK)-like state with superior anti-tumor potential. Temporal induction of Stat5 activity in Tex cells using an orthogonal IL-2:IL2Rß-pair fostered Texint cell accumulation, particularly upon PD-L1 blockade. Re-engaging Stat5 also partially reprogrammed the epigenetic landscape of exhaustion and restored polyfunctionality. These data highlight therapeutic opportunities of manipulating the IL-2-Stat5 axis to rewire Tex cells toward more durably protective states.

A statistical framework for differential pseudotime analysis with multiple single-cell RNA-seq samples.

Pseudotime analysis with single-cell RNA-sequencing (scRNA-seq) data has been widely used to study dynamic gene regulatory programs along continuous biological processes. While many methods have been developed to infer the pseudotemporal trajectories of cells within a biological sample, it remains a challenge to compare pseudotemporal patterns with multiple samples (or replicates) across different experimental conditions. Here, we introduce Lamian, a comprehensive and statistically-rigorous computational framework for differential multi-sample pseudotime analysis. Lamian can be used to identify changes in a biological process associated with sample covariates, such as different biological conditions while adjusting for batch effects, and to detect changes in gene expression, cell density, and topology of a pseudotemporal trajectory. Unlike existing methods that ignore sample variability, Lamian draws statistical inference after accounting for cross-sample variability and hence substantially reduces sample-specific false discoveries that are not generalizable to new samples. Using both real scRNA-seq and simulation data, including an analysis of differential immune response programs between COVID-19 patients with different disease severity levels, we demonstrate the advantages of Lamian in decoding cellular gene expression programs in continuous biological processes.

Deep phenotyping of the lipidomic response in COVID-19 and non-COVID-19 sepsis.

BACKGROUND: Lipids may influence cellular penetrance by viral pathogens and the immune response that they evoke. We deeply phenotyped the lipidomic response to SARs-CoV-2 and compared that with infection with other pathogens in patients admitted with acute respiratory distress syndrome to an intensive care unit (ICU). METHODS: Mass spectrometry was used to characterise lipids and relate them to proteins, peripheral cell immunotypes and disease severity. RESULTS: Circulating phospholipases (sPLA2, cPLA2 (PLA2G4A) and PLA2G2D) were elevated on admission in all ICU groups. Cyclooxygenase, lipoxygenase and epoxygenase products of arachidonic acid (AA) were elevated in all ICU groups compared with controls. sPLA2 predicted severity in COVID-19 and correlated with TxA2, LTE4 and the isoprostane, iPF2α-III, while PLA2G2D correlated with LTE4. The elevation in PGD2, like PGI2 and 12-HETE, exhibited relative specificity for COVID-19 and correlated with sPLA2 and the interleukin-13 receptor to drive lymphopenia, a marker of disease severity. Pro-inflammatory eicosanoids remained correlated with severity in COVID-19 28 days after admission. Amongst non-COVID ICU patients, elevations in 5- and 15-HETE and 9- and 13-HODE reflected viral rather than bacterial disease. Linoleic acid (LA) binds directly to SARS-CoV-2 and both LA and its di-HOME products reflected disease severity in COVID-19. In healthy marines, these lipids rose with seroconversion. Eicosanoids linked variably to the peripheral cellular immune response. PGE2, TxA2 and LTE4 correlated with T cell activation, as did PGD2 with non-B non-T cell activation. In COVID-19, LPS stimulated peripheral blood mononuclear cell PGF2α correlated with memory T cells, dendritic and NK cells while LA and DiHOMEs correlated with exhausted T cells. Three high abundance lipids - ChoE 18:3, LPC-O-16:0 and PC-O-30:0 - were altered specifically in COVID. LPC-O-16:0 was strongly correlated with T helper follicular cell activation and all three negatively correlated with multi-omic inflammatory pathways and disease severity. CONCLUSIONS: A broad based lipidomic storm is a predictor of poor prognosis in ARDS. Alterations in sPLA2, PGD2 and 12-HETE and the high abundance lipids, ChoE 18:3, LPC-O-16:0 and PC-O-30:0 exhibit relative specificity for COVID-19 amongst such patients and correlate with the inflammatory response to link to disease severity.