CD38 marks the exhausted CD8+ tissue-resident memory T cells in hepatocellular carcinoma.

Introduction: Despite recent advances in immunotherapy for hepatocellular carcinoma (HCC), the overall modest response rate underscores the need for a better understanding of the tumor microenvironment (TME) of HCC. We have previously shown that CD38 is widely expressed on tumor-infiltrating leukocytes (TILs), predominantly on CD3+ T cells and monocytes. However, its specific role in the HCC TME remains unclear. Methods: In this current study, we used cytometry time-of-flight (CyTOF), bulk RNA sequencing on sorted T cells, and single-cell RNA (scRNA) sequencing to interrogate expression of CD38 and its correlation with T cell exhaustion in HCC samples. We also employed multiplex immunohistochemistry (mIHC) for validating our findings. Results: From CyTOF analysis, we compared the immune composition of CD38-expressing leukocytes in TILs, non-tumor tissue-infiltrating leukocytes (NIL), and peripheral blood mononuclear cells (PBMC). We identified CD8+ T cells as the dominant CD38-expressing TILs and found that CD38 expression was significantly higher in CD8+ TRM in TILs than in NILs. Furthermore, through transcriptomic analysis on sorted CD8+ TRM from HCC tumors, we observed a higher expression of CD38 along with T cell exhaustion genes, including PDCD1 and CTLA4, compared to the circulating memory CD8 T cells from PBMC. This was validated by scRNA sequencing that revealed co-expression of CD38 with PDCD1, CTLA4, and ITGAE (CD103) in T cells from HCC tumors. The protein co-expression of CD38 and PD-1 on CD8+ T cells was further demonstrated by mIHC on HCC FFPE tissues, marking CD38 as a T cell co-exhaustion marker in HCC. Lastly, the higher proportions of CD38+PD-1+ CD8+ T cells and CD38+PD-1+ TRM were significantly associated with the higher histopathological grades of HCC, indicating its role in the aggressiveness of the disease. Conclusion: Taken together, the concurrent expression of CD38 with exhaustion markers on CD8+ TRM underpins its role as a key marker of T cell exhaustion and a potential therapeutic target for restoring cytotoxic T cell function in HCC.

IFNγ-IL-17+ CD8 T cells contribute to immunosuppression and tumor progression in human hepatocellular carcinoma.

IL-17-producing CD8 (Tc17) T cells have been shown to play an important role in infection and chronic inflammation, however their implications in hepatocellular carcinoma (HCC) remain elusive. In this study, we performed cytometry by time-of-flight (CyTOF) and revealed the distinctive immunological phenotypes of two IFNγ+ and IFNγ- Tc17 subsets that were preferentially enriched in human HCC. Single-cell RNA-sequencing analysis further revealed regulatory circuits governing the different phenotypes of these Tc17 subsets. In particular, we discovered that IFNγ- Tc17 subset demonstrated pro-tumoral characteristics and expressed higher levels of CCL20. This corresponded to increased tumor infiltration of T regulatory cells (Treg) validated by immunohistochemistry in another independent HCC cohort, demonstrating the immunosuppressive functions of IFNγ- Tc17 subset. Most importantly, higher intra-tumoral proportions of IFNγ- Tc17 were associated with poorer prognosis in patients with HCC and this was further validated in The Cancer Genome Atlas (TCGA) HCC cohort. Taken together, this compendium of transcriptomic and proteomic data of Tc17 subsets sheds light on the immunosuppressive phenotypes of IFNγ- Tc17 and its implications in HCC progression.

Single-cell transcriptomics and surface epitope detection in human brain epileptic lesions identifies pro-inflammatory signaling.

Epileptogenic triggers are multifactorial and not well understood. Here we aimed to address the hypothesis that inappropriate pro-inflammatory mechanisms contribute to the pathogenesis of refractory epilepsy (non-responsiveness to antiepileptic drugs) in human patients. We used single-cell cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to reveal the immunotranscriptome of surgically resected epileptic lesion tissues. Our approach uncovered a pro-inflammatory microenvironment, including extensive activation of microglia and infiltration of other pro-inflammatory immune cells. These findings were supported by ligand-receptor (LR) interactome analysis, which demonstrated potential mechanisms of infiltration and evidence of direct physical interactions between microglia and T cells. Together, these data provide insight into the immune microenvironment in epileptic tissue, which may aid the development of new therapeutics.

Uncoupling immune trajectories of response and adverse events from anti-PD-1 immunotherapy in hepatocellular carcinoma.

BACKGROUND & AIMS: While immune checkpoint blockade (ICB) has shown promise in patients with hepatocellular carcinoma (HCC), it is associated with modest response rates and immune-related adverse events (irAEs) are common. In this study, we aimed to decipher immune trajectories and mechanisms of response and/or irAEs in patients with HCC receiving anti-programmed cell death 1 (anti-PD-1) therapy. METHODS: Pre- and on-treatment peripheral blood samples (n = 60) obtained from 32 patients with HCC (Singapore cohort) were analysed by cytometry by time-of-flight and single-cell RNA sequencing, with flow cytometric validation in an independent Korean cohort (n = 29). Mechanistic validation was conducted by bulk RNA sequencing of 20 pre- and on-treatment tumour biopsies and using a murine HCC model treated with different immunotherapeutic combinations. RESULTS: Single-cell analyses identified CXCR3+CD8+ effector memory T (TEM) cells and CD11c+ antigen-presenting cells (APC) as associated with response (p = 0.0004 and 0.0255, respectively), progression-free survival (p = 0.00079 and 0.0015, respectively), and irAEs (p = 0.0034 and 0.0125, respectively) in anti-PD-1-treated patients with HCC. Type-1 conventional dendritic cells were identified as the specific APC associated with response, while 2 immunosuppressive CD14+ myeloid clusters were linked to reduced irAEs. Further analyses of CXCR3+CD8+ TEM cells showed cell-cell interactions specific to response vs. irAEs, from which the anti-PD-1 and anti-TNFR2 combination was harnessed to uncouple these effects, resulting in enhanced response without increased irAEs in a murine HCC model. CONCLUSIONS: This study identifies early predictors of clinical response to anti-PD-1 ICB in patients with HCC and offers mechanistic insights into the immune trajectories of these immune subsets at the interface between response and toxicity. We also propose a new combination immunotherapy for HCC to enhance response without exacerbating irAEs. CLINICAL TRIAL NUMBER: NCT03695952. LAY SUMMARY: Response rates to immune checkpoint blockade (ICB) treatment in hepatocellular carcinoma (HCC) remain modest and adverse events are common. Herein, we identified early predictors of response and gained an in-depth understanding of the immunological mechanisms behind response and adverse events in patients with HCC treated with ICB. We also proposed a new combination immunotherapy for HCC that enhances response without exacerbating adverse events.

Pro-Inflammatory Derangement of the Immuno-Interactome in Heart Failure.

Chronic heart failure (HF) is a syndrome of heterogeneous etiology associated with multiple co-morbidities. Inflammation is increasingly recognized as a key contributor to the pathophysiology of HF. Heterogeneity and lack of data on the immune mechanism(s) contributing to HF may partially underlie the failure of clinical trials targeting inflammatory mediators. We studied the Immunome in HF cohort using mass cytometry and used data-driven systems immunology approach to discover and characterize modulated immune cell subsets from peripheral blood. We showed cytotoxic and inflammatory innate lymphoid and myeloid cells were expanded in HF patients compared to healthy controls. Network analysis showed highly modular and centralized immune cell architecture in healthy control immune cell network. In contrast, the HF immune cell network showed greater inter-cellular communication and less modular structure. Furthermore, we found, as an immune mechanism specific to HF with preserved ejection fraction (HFpEF), an increase in inflammatory MAIT and CD4 T cell subsets.

Robust neutralizing antibody response to SARS-CoV-2 mRNA vaccination in adolescents and young adults with childhood-onset rheumatic diseases.

OBJECTIVES: Immunogenicity to the SARS-CoV-2 mRNA vaccines in adolescents and young adults (AYA) with childhood-onset rheumatic diseases (cRD) is unknown. We aimed to evaluate the humoral immunogenicity and safety of the vaccines in our AYA with cRD. METHODS: A monocentric observational study with 159 AYA (50.3% female and 70.4% Chinese). Humoral immunogenicity was assessed at 2-3 and 4-6 weeks following first and second vaccination by cPass™ SARS-CoV-2 Neutralization Antibody Assay. Inhibition signal of ≥30% defined the cut-off for positive detection of the SARS-CoV-2 neutralizing antibodies. Vaccine safety and disease activity were assessed within 6 weeks after second vaccination. RESULTS: A total of 64.9% and 99.1% of 159 patients (median age: 16.9, IQR: 14.7-19.5) mounted positive SARS-CoV-2 neutralizing responses after first and second vaccination, respectively. Most patients (89.8%) had ≥90% inhibition signal after second vaccination. Methotrexate and mycophenolate mofetil increased the risk associated with negative cPass neutralization responses following the first vaccination. Holding both medications after each vaccination did not affect immunogenicity. There was no symptomatic COVID-19 infection. Local reaction remained the most common (23.3-25.2%) adverse event, without serious complication. Two and seven patients flared following the first and second vaccination, respectively. Subgroup analyses of the 12-18-year-old cohort did not show any differences in vaccine efficacy, predictors of poor response and general safety, but higher proportion of disease flares. CONCLUSIONS: SARS-CoV-2 mRNA vaccines were efficacious after the two-dose regimen in almost all AYA with cRD without serious adverse event. The rate of disease flare observed is 4.4% after the second mRNA vaccine dose.

Hypoxia-driven immunosuppression by Treg and type-2 conventional dendritic cells in HCC.

BACKGROUND AND AIMS: Hypoxia is one of the central players in shaping the immune context of the tumor microenvironment (TME). However, the complex interplay between immune cell infiltrates within the hypoxic TME of HCC remains to be elucidated. APPROACH AND RESULTS: We analyzed the immune landscapes of hypoxia-low and hypoxia-high tumor regions using cytometry by time of light, immunohistochemistry, and transcriptomic analyses. The mechanisms of immunosuppression in immune subsets of interest were further explored using in vitro hypoxia assays. Regulatory T cells (Tregs) and a number of immunosuppressive myeloid subsets, including M2 macrophages and human leukocyte antigen-DR isotype (HLA-DRlo ) type 2 conventional dendritic cell (cDC2), were found to be significantly enriched in hypoxia-high tumor regions. On the other hand, the abundance of active granzyme Bhi PD-1lo CD8+ T cells in hypoxia-low tumor regions implied a relatively active immune landscape compared with hypoxia-high regions. The up-regulation of cancer-associated genes in the tumor tissues and immunosuppressive genes in the tumor-infiltrating leukocytes supported a highly pro-tumorigenic network in hypoxic HCC. Chemokine genes such as CCL20 (C-C motif chemokine ligand 20) and CXCL5 (C-X-C motif chemokine ligand 5) were associated with recruitment of both Tregs and HLA-DRlo cDC2 to hypoxia-high microenvironments. The interaction between Tregs and cDC2 under a hypoxic TME resulted in a loss of antigen-presenting HLA-DR on cDC2. CONCLUSIONS: We uncovered the unique immunosuppressive landscapes and identified key immune subsets enriched in hypoxic HCC. In particular, we identified a potential Treg-mediated immunosuppression through interaction with a cDC2 subset in HCC that could be exploited for immunotherapies.

A Virus-Specific Immune Rheostat in the Immunome of Patients Recovering From Mild COVID-19.

An accurate depiction of the convalescent COVID-19 immunome will help delineate the immunological milieu crucial for disease resolution and protection. Using mass cytometry, we characterized the immune architecture in patients recovering from mild COVID-19. We identified a virus-specific immune rheostat composed of an effector T (Teff) cell recall response that is balanced by the enrichment of a highly specialized regulatory T (Treg) cell subset. Both components were reactive against a peptide pool covering the receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein. We also observed expansion of IFNγ+ memory CD4+ T cells and virus-specific follicular helper T (TFH) cells. Overall, these findings pinpoint critical immune effector and regulatory mechanisms essential for a potent, yet harmless resolution of COVID-19 infection.

Neutralizing Activity and SARS-CoV-2 Vaccine mRNA Persistence in Serum and Breastmilk After BNT162b2 Vaccination in Lactating Women.

Background: There is limited information on the functional neutralizing capabilities of breastmilk SARS-CoV-2-specific antibodies and the potential adulteration of breastmilk with vaccine mRNA after SARS-CoV-2 mRNA vaccination. Methods: We conducted a prospective cohort study of lactating healthcare workers who received the BNT162b2 vaccine and their infants. The presence of SARS-CoV-2 neutralizing antibodies, antibody isotypes (IgG, IgA, IgM) and intact mRNA in serum and breastmilk was evaluated at multiple time points using a surrogate neutralizing assay, ELISA, and PCR, over a 6 week period of the two-dose vaccination given 21 days apart. Results: Thirty-five lactating mothers, median age 34 years (IQR 32-36), were included. All had detectable neutralizing antibodies in the serum immediately before dose 2, with significant increase in neutralizing antibody levels 7 days after this dose [median 168.4 IU/ml (IQR 100.7-288.5) compared to 2753.0 IU/ml (IQR 1627.0-4712.0), p <0.001]. Through the two vaccine doses, all mothers had detectable IgG1, IgA and IgM isotypes in their serum, with a notable increase in all three antibody isotypes after dose 2, especially IgG1 levels. Neutralizing antibodies were detected in majority of breastmilk samples a week after dose 2 [median 13.4 IU/ml (IQR 7.0-28.7)], with persistence of these antibodies up to 3 weeks after. Post the second vaccine dose, all (35/35, 100%) mothers had detectable breastmilk SARS-CoV-2 spike RBD-specific IgG1 and IgA antibody and 32/35 (88.6%) mothers with IgM. Transient, low intact vaccine mRNA levels was detected in 20/74 (27%) serum samples from 21 mothers, and 5/309 (2%) breastmilk samples from 4 mothers within 1 weeks of vaccine dose. Five infants, median age 8 months (IQR 7-16), were also recruited - none had detectable neutralizing antibodies or vaccine mRNA in their serum. Conclusion: Majority of lactating mothers had detectable SARS-CoV-2 antibody isotypes and neutralizing antibodies in serum and breastmilk, especially after dose 2 of BNT162b2 vaccination. Transient, low levels of vaccine mRNA were detected in the serum of vaccinated mothers with occasional transfer to their breastmilk, but we did not detect evidence of infant sensitization. Importantly, the presence of breastmilk neutralising antibodies likely provides a foundation for passive immunisation of the breastmilk-fed infant.

Systemic Sclerosis Perturbs the Architecture of the Immunome.

Systemic sclerosis (SSc) is an autoimmune disease characterized by excessive fibrosis of skin and internal organs, and vascular dysfunction. Association of T and B cell subsets has been reported in SSc; however, there is lack of systematic studies of functional relations between immune cell subsets in this disease. This lack of mechanistic knowledge hampers targeted intervention. In the current study we sought to determine differential immune cell composition and their interactions in peripheral blood of SSc patients. Mononuclear cells from blood of SSc patients (n = 20) and healthy controls (n = 10) were analyzed by mass cytometry using a 36-marker (cell surface and intracellular) panel. Transcriptome analysis (m-RNA sequencing) was performed on sorted T and B cell subsets. Unsupervised clustering analysis revealed significant differences in the frequencies of T and B cell subsets in patients. Correlation network analysis highlighted an overall dysregulated immune architecture coupled with domination of inflammatory senescent T cell modules in SSc patients. Transcriptome analysis of sorted immune cells revealed an activated phenotype of CD4 and mucosal associated invariant T (MAIT) cells in patients, accompanied by increased expression of inhibitory molecules, reminiscent of phenotype exhibited by functionally adapted, exhausted T cells in response to chronic stimulation. Overall, this study provides an in-depth analysis of the systemic immunome in SSc, highlighting the potential pathogenic role of inflammation and chronic stimulation-mediated "functional adaptation" of immune cells.

Publisher Correction: The Extended Polydimensional Immunome Characterization (EPIC) web-based reference and discovery tool for cytometry data.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Immunological Hallmarks for Clinical Response to BCG in Bladder Cancer.

Intravesical Bacillus Calmette-Guerin (BCG) is an effective immunotherapy for non-muscle invasive bladder cancer (NMIBC). However, recurrence and progression remain frequent warranting deeper insights into its mechanism. We herein comprehensively profiled blood and tissues obtained from NMIBC patients before, during and after BCG treatment using cytometry by time-of-flight (CyTOF) and RNA sequencing to identify the key immune subsets crucial for anti-tumor activity. We observed the temporal changes of peripheral immune subsets including NKT cells, central memory CD4+ T cells, CD8+ T cells and regulatory T cells (Treg) during the course of BCG. Gene expression analysis revealed enriched immune pathways involving in T cell activation and chemotaxis, as well as a more diversified T cell receptor repertoire in post-BCG tissues. Moreover, tissue multiplexed-immunofluorescence (mIF) showed baseline densities of non-Treg and CD8+PD-1+ T cells were predictive of response and better recurrence-free survival after BCG. Remarkably, post-BCG tissues from responders were found to be infiltrated with more active CD8+PD-1- T cells and non-Treg CD4+FOXP3- T cells; but increased exhausted CD8+PD-1+ T cells were found in non-responders. Taken together, we identified predictive biomarkers for response and uncovered the post-treatment expansion of exhausted PD-1+CD8+ T cells as key to BCG resistance, which could potentially be restored by combining with anti-PD-1 immunotherapy.

Immunome perturbation is present in patients with juvenile idiopathic arthritis who are in remission and will relapse upon anti-TNFα withdrawal.

OBJECTIVES: Biologics treatment with antitumour necrosis factor alpha (TNFα) is efficacious in patients with juvenile idiopathic arthritis (JIA). Despite displaying clinical inactivity during treatment, many patients will flare on cessation of therapy. The inability to definitively discriminate patients who will relapse or continue to remain in remission after therapy withdrawal is currently a major unmet medical need. CD4 T cells have been implicated in active disease, yet how they contribute to disease persistence despite treatment is unknown. METHODS: We interrogated the circulatory reservoir of CD4+ immune subsets at the single-cell resolution with mass cytometry (cytometry by time of flight) of patients with JIA (n=20) who displayed continuous clinical inactivity for at least 6 months with anti-TNFα and were subsequently withdrawn from therapy for 8 months, and scored as relapse or remission. These patients were examined prior to therapy withdrawal for putative subsets that could discriminate relapse from remission. We verified on a separate JIA cohort (n=16) the dysregulation of these circulatory subsets 8 months into therapy withdrawal. The immunological transcriptomic signature of CD4 memory in relapse/remission patients was examined with NanoString. RESULTS: An inflammatory memory subset of CD3+CD4+CD45RA-TNFα+ T cells deficient in immune checkpoints (PD1-CD152-) was present in relapse patients prior to therapy withdrawal. Transcriptomic profiling reveals divergence between relapse and remission patients in disease-centric pathways involving (1) T-cell receptor activation, (2) apoptosis, (3) TNFα, (4) nuclear factor-kappa B and (5) mitogen-activated protein kinase signalling. CONCLUSIONS: A unique discriminatory immunomic and transcriptomic signature is associated with relapse patients and may explain how relapse occurs.

Immune activation underlies a sustained clinical response to Yttrium-90 radioembolisation in hepatocellular carcinoma.

OBJECTIVES: Yttrium-90 (Y90)-radioembolisation (RE) significantly regresses locally advanced hepatocellular carcinoma and delays disease progression. The current study is designed to deeply interrogate the immunological impact of Y90-RE, which elicits a sustained therapeutic response. DESIGN: Time-of-flight mass cytometry and next-generation sequencing (NGS) were used to analyse the immune landscapes of tumour-infiltrating leucocytes (TILs), tumour tissues and peripheral blood mononuclear cells (PBMCs) at different time points before and after Y90-RE. RESULTS: TILs isolated after Y90-RE exhibited signs of local immune activation: higher expression of granzyme B (GB) and infiltration of CD8+ T cells, CD56+ NK cells and CD8+ CD56+ NKT cells. NGS confirmed the upregulation of genes involved in innate and adaptive immune activation in Y90-RE-treated tumours. Chemotactic pathways involving CCL5 and CXCL16 correlated with the recruitment of activated GB+CD8+ T cells to the Y90-RE-treated tumours. When comparing PBMCs before and after Y90-RE, we observed an increase in tumour necrosis factor-α on both the CD8+ and CD4+ T cells as well as an increase in percentage of antigen-presenting cells after Y90-RE, implying a systemic immune activation. Interestingly, a high percentage of PD-1+/Tim-3+CD8+ T cells coexpressing the homing receptors CCR5 and CXCR6 denoted Y90-RE responders. A prediction model was also built to identify sustained responders to Y90-RE based on the immune profiles from pretreatment PBMCs. CONCLUSION: High-dimensional analysis of tumour and systemic immune landscapes identified local and systemic immune activation that corresponded to the sustained response to Y90-RE. Potential biomarkers associated with a positive clinical response were identified and a prediction model was built to identify sustained responders prior to treatment.

Multidimensional analyses reveal distinct immune microenvironment in hepatitis B virus-related hepatocellular carcinoma.

BACKGROUND AND AIMS: Chronic inflammation induced by chronic hepatitis B virus (HBV) infection increases the risk of hepatocellular carcinoma (HCC). However, little is known about the immune landscape of HBV-related HCC and its influence on the design of effective cancer immunotherapeutics. METHODS: We interrogated the immune microenvironments of HBV-related HCC and non-viral-related HCC using immunohistochemistry and cytometry by time-of-flight (CyTOF). On identifying unique immune subsets enriched in HBV-related HCC, we further interrogated their phenotypes and functions using next-generation sequencing (NGS) and in vitro T-cell proliferation assays. RESULTS: In-depth interrogation of the immune landscapes showed that regulatory T cells (TREG) and CD8+ resident memory T cells (TRM) were enriched in HBV-related HCC, whereas Tim-3+CD8+ T cells and CD244+ natural killer cells were enriched in non-viral-related HCC. NGS of isolated TREG and TRM from HBV-related HCC and non-viral-related HCC identified distinct functional signatures associated with T-cell receptor signalling, T-cell costimulation, antigen presentation and programmed cell death protein 1 (PD-1) signalling. TREG and TRM from HBV-related HCC expressed more PD-1 and were functionally more suppressive and exhausted than those from non-virus-related HCC. Furthermore, immunosuppression by PD-1+ TREG could be reversed with anti-PD-1 blockade. Using multiplexed tissue immunofluorescence, we further demonstrated that TREG and TRM contributed to overall patient survival: TREG were associated with a poor prognosis and TRM were associated with a good prognosis in HCC. CONCLUSION: We have shown that the HBV-related HCC microenvironment is more immunosuppressive and exhausted than the non-viral-related HCC microenvironment. Such in-depth understanding has important implications in disease management and appropriate application of immunotherapeutics.

Molecular mechanisms of autophagic memory in pathogenic T cells in human arthritis.

T-cell resilience is critical to the immune pathogenesis of human autoimmune arthritis. Autophagy is essential for memory T cell generation and associated with pathogenesis in rheumatoid arthritis (RA). Our aim here was to delineate the role and molecular mechanism of autophagy in resilience and persistence of pathogenic T cells from autoimmune arthritis. We demonstrated "Autophagic memory" as elevated autophagy levels in CD4+ memory T cells compared to CD4+ naive T cells and in Jurkat Human T cell line trained with starvation stress. We then showed increased levels of autophagy in pathogenic CD4+ T cells subsets from autoimmune arthritis patients. Using RNA-sequencing, transcription factor gene regulatory network and methylation analyses we identified MYC as a key regulator of autophagic memory. We validated MYC levels using qPCR and further demonstrated that inhibiting MYC increased autophagy. The present study proposes the novel concept of autophagic memory and suggests that autophagic memory confers metabolic advantage to pathogenic T cells from arthritis and supports its resilience and long term survival. Particularly, suppression of MYC imparted the heightened autophagy levels in pathogenic T cells. These studies have a direct translational valency as they identify autophagy and its metabolic controllers as a novel therapeutic target.

A systematic approach to the development of a safe live attenuated Zika vaccine.

Zika virus (ZIKV) is a flavivirus that can cause congenital disease and requires development of an effective long-term preventative strategy. A replicative ZIKV vaccine with properties similar to the yellow fever 17D (YF17D) live-attenuated vaccine (LAV) would be advantageous, as a single dose of YF17D produces lifelong immunity. However, a replicative ZIKV vaccine must also be safe from causing persistent organ infections. Here we report an approach to ZIKV LAV development. We identify a ZIKV variant that produces small plaques due to interferon (IFN)-restricted viral propagation and displays attenuated infection of endothelial cells. We show that these properties collectively reduce the risk of organ infections and vertical transmission in a mouse model but remain sufficiently immunogenic to prevent wild-type ZIKV infection. Our findings suggest a strategy for the development of a safe but efficacious ZIKV LAV.

Early molecular correlates of adverse events following yellow fever vaccination.

The innate immune response shapes the development of adaptive immunity following infections and vaccination. However, it can also induce symptoms such as fever and myalgia, leading to the possibility that the molecular basis of immunogenicity and reactogenicity of vaccination are inseparably linked. To test this possibility, we used the yellow fever live-attenuated vaccine (YFLAV) as a model to study the molecular correlates of reactogenicity or adverse events (AEs). We analyzed the outcome of 68 adults who completed a YFLAV clinical trial, of which 43 (63.2%) reported systemic AEs. Through whole-genome profiling of blood collected before and after YFLAV dosing, we observed that activation of innate immune genes at day 1, but not day 3 after vaccination, was directly correlated with AEs. These findings contrast with the gene expression profile at day 3 that we and others have previously shown to be correlated with immunogenicity. We conclude that although the innate immune response is a double-edged sword, its expression that induces AEs is temporally distinct from that which engenders robust immunity. The use of genomic profiling thus provides molecular insights into the biology of AEs that potentially forms a basis for the development of safer vaccines.

Delineation of an immunosuppressive gradient in hepatocellular carcinoma using high-dimensional proteomic and transcriptomic analyses.

The recent development of immunotherapy as a cancer treatment has proved effective over recent years, but the precise dynamics between the tumor microenvironment (TME), nontumor microenvironment (NTME), and the systemic immune system remain elusive. Here, we interrogated these compartments in hepatocellular carcinoma (HCC) using high-dimensional proteomic and transcriptomic analyses. By time-of-flight mass cytometry, we found that the TME was enriched in regulatory T cells (Tregs), tissue resident memory CD8+ T cells (TRMs), resident natural killer cells (NKRs), and tumor-associated macrophages (TAMs). This finding was also validated with immunofluorescence staining on Foxp3+CD4+ and PD-1+CD8+ T cells. Interestingly, Tregs and TRMs isolated from the TME expressed multiple markers for T-cell exhaustion, including PD-1, Lag-3, and Tim-3 compared with Tregs and TRMs isolated from the NTME. We found PD-1+ TRMs were the predominant T-cell subset responsive to anti-PD-1 treatment and significantly reduced in number with increasing HCC tumor progression. Furthermore, T-bet was identified as a key transcription factor, negatively correlated with PD-1 expression on memory CD8+ T cells, and the PD-1:T-bet ratio increased upon exposure to tumor antigens. Finally, transcriptomic analysis of tumor and adjacent nontumor tissues identified a chemotactic gradient for recruitment of TAMs and NKRs via CXCR3/CXCL10 and CCR6/CCL20 pathways, respectively. Taken together, these data confirm the existence of an immunosuppressive gradient across the TME, NTME, and peripheral blood in primary HCC that manipulates the activation status of tumor-infiltrating leukocytes and renders them immunocompromised against tumor cells. By understanding the immunologic composition of this gradient, more effective immunotherapeutics for HCC may be designed.