Trisomy 21 increases the risk of severe outcomes and mortality in hospitalized individuals with Down syndrome (DS) following SARS-CoV-2 infection. Using data from the Brazilian Epidemiological Surveillance Information System Influenza (SIVEP-Gripe), we analyzed 102,767 hospitalized COVID-19 patients (1,115 DS and 101,652 non-DS, NDS). DS patients had a higher prevalence of comorbidities and required ventilatory support, ICU admission, and intubation more frequently than NDS patients (p < 0.001). Mortality was 4.5 times higher in DS patients aged 0-30 years (26.3% vs. 5.9%, p < 0.001) and remained 2.22 times higher after adjusting for comorbidities. DS patients over 30 years also exhibited a 22% increase in mortality (PR 1.22, p < 0.001). Gene expression analysis of pre-pandemic monocytes and T cells from DS individuals revealed upregulated pathways linked to SARS-CoV-2 infection, including interferon signaling and cytokine interactions. This baseline immune dysregulation may contribute to severe COVID-19 outcomes in DS patients. Identifying these altered pathways could inform targeted therapeutic strategies to improve immune homeostasis and clinical outcomes. To the best of our knowledge, this is the first study integrating nationwide clinical outcomes with pre-pandemic immune transcriptomic data to mechanistically explain the heightened COVID-19 severity in individuals with DS.

Lymph nodes (LNs) constitute a key anatomical sanctuary for HIV. Follicular helper T (Tfh) cells expand early upon infection and represent a principal cellular target for initial viral seeding. Here, we identified the transcription factor BCL6, a Tfh-lineage defining marker, as central in favoring the infection of Tfh cells in LNs during the untreated phase in humans, and for the persistence of the reservoir during ART in non-human primates. In situ and ex vivo analyses of LN from people with HIV (PWH) in absence of antiretroviral therapy (ART) revealed preferential enrichment of viral RNA, total HIV DNA, and intact proviruses within BCL6hi Tfh cells, which also presented significantly lower expression of proteins with antiviral functions (IRF7, MX1, APOBEC3G, pSTAT1). In vitro genetic (genome-wide CRISPR knockouts) and pharmacologic perturbations confirmed that BCL6 enhances the cellular permissiveness of Tfh cells to HIV infection. IL-10 and TGF-β were enriched in LNs from people without HIV (PWoH), and cooperatively induced bona fide BCL6hi Tfh differentiation in vitro, with repressed antiviral pathways. IL-10 and TGF-β blockade limited Tfh differentiation, confirming their contribution to Tfh and LN biology. Human Single Nucleotide Polymorphisms (SNPs) in proximity to genes of the IL-10 and TGF-β pathways were enriched in PWH who controls viremia spontaneously (HIV elite controllers). Importantly, in vivo downmodulation of IL-10 and TGF-β signaling pathways in ART-treated SIV-infected macaques, by using anti–IL-10 and anti–PD-1 therapy, led to reduced frequencies of LN BCL6+ Tfh cells. These Tfh cells expressed significantly higher expression of antiviral machineries, similar to gene signatures found in HIV elite controllers, and resulted in significantly lower SIV reservoir size in LNs. This data highlights that the modulation of the IL-10/TGF-β/BCL6 axis is relevant at early stages upon infection, but also during ART, after the HIV reservoir is already established. In both scenarios it results in higher antiviral machinery and lower HIV seeding and reservoir sizes. Thus, the modulation of these pathways in vivo has potential to alter Tfh biology in LNs leading to HIV reservoir decay, contributing to HIV cure strategies.

Bile acids (BAs) a complex family of immunometabolites can shape myeloid cell function; their role in antiviral immunity and clinical outcomes in human infection remains unclear. We identified an early plasma specific BA signature that distinguished moderate from severe COVID-19 trajectories (M vs. SCHP) in >1,100 hospitalized patients. Single-cell RNA and ATAC sequencing of PBMCs identified a TGR5⁺ monocyte subset in MCHP characterized by increased chromatin accessibility and expression of interferon-responsive antiviral and MHC class II pathways, and reduced IL-10 signaling. External datasets confirmed enrichment of this program in MCHP and healthy donors. In TGR5⁺ monocytes of MCHP, these BAs synergized with IFN-γ to enhance STAT1 activation, ERK/CREB1 signaling, and H3K27ac; independently promoting trained immunity recall responses. We reveal a previously unrecognized antiviral axis whereby these specific BAs activate TGR5 to drive transcriptional and epigenetic programs associated with upregulation of antiviral responses, trained immunity, and favorable clinical outcomes in COVID-19

Immunotherapeutic approaches to eliminate latently HIV-infected cells are focused on the adaptive immune system. Herein we provide mechanistic evidence for a molecular cascade characterized by epigenetic reprogramming of innate myeloid cells and CD4 T cells. The coordinate regulation and gene expression mediated by transcription factors (TFs) IRF3, IRF7, STAT1 and C/EBPβ versus AP-1, promoted the development of innate antiviral immunity in these cells which was associated with control of viral load and decay of cell associated viral DNA (CA-vDNA) following analytical treatment interruption (ATI) in SIV-infected rhesus macaques (RMs) treated with anti-IL-10 and anti-PD-1. The prevalence of TGF-β/SMAD signaling in a subset of combo-treated RMs with high CA-vDNA (CA-vDNAhi) suppressed this antiviral activity through histone deacetylases, including HDAC11, as the latter reduced chromatin accessibility of IRFs and STATs and impeded their antiviral functions. The addition of HDAC inhibitors in vitro restored antiviral response in the presence of TGF-β. Induction of IL-6, a target gene of C/EBPβ, in CA-vDNAlo RMs, amplified the antiviral network through IRF9, a transcription factor upstream of IRF7. We identified a similar molecular cascade in HIV elite controllers, who maintain low to undetectable viremia and small viral reservoirs without treatment. These data highlight the importance of epigenetic regulation of the host in shaping innate antiviral immune responses that control viral rebound following ATI and reduce the viral reservoir, providing insight into potential strategies for HIV cure interventions.

Immunotherapeutic approaches to eliminate latently HIV-infected cells are focused on the adaptive immune system. Herein we provide mechanistic evidence for a molecular cascade highlighted by epigenetic reprogramming of innate myeloid cells mediated by specific transcription factors (IRF-3, IRF-7, STAT-1, and C/EBPβ). This cascade promotes the development of innate antiviral immunity that triggers viral load control and viral DNA (vDNA) decay post-ATI in SIV-infected rhesus macaques (RMs) treated with anti-IL-10 and anti-PD-1. The balance between the antiviral signaling driven by IRFs, STATs, and C/EBPβ and the pro-inflammatory response mediated by AP-1 triggered the decay of vDNA levels in lymph nodes. TGF-β/SMAD signaling suppressed this antiviral activity through HDAC11, which blocked the chromatin accessibility of IRFs/STATs and impeded their antiviral functions. HDAC inhibitors restored this antiviral response in the presence of TGF-β. IL-6 induction, a target of C/EBPβ, amplified the antiviral network through IRF9, a TF upstream of IRF7, a feature specific of RMs with lower vDNA levels. HIV elite controllers, who maintain undetectable viremia and small viral reservoirs without treatment showed similar molecular cascade. In summary, our data highlights the importance of epigenetic regulation in shaping innate antiviral immune response that controls viral rebound and reduces the viral reservoir, providing insight into potential strategies for HIV cure interventions. R37AI141258 P01AI178376 UM1AI164561 Viral Immunology (VIR)

T cells play a pivotal role in the immune system, relying on their somatically rearranged T cell receptor (TCR) to recognize peptide-MHC complexes. A comprehensive and extensively used set of monoclonal antibodies (mAbs) against TCR variable regions was generated in the previous century. The separate identification of mAb-specific TCR-V proteins and TRV genes has resulted in multiple nomenclatures, making their relationships unclear. To formally re-establish this link and determine patterns of reactivity within TRV subfamilies, we sorted T cells from C57BL/6 mice positive for any one of a panel of 22 anti-V mAbs and determined their TRV genes by single-cell TCRseq. RNAseq data revealed consistently higher expression of repeated elements from the ERV1-family LTR RLTR6Mm (mapping to Gm20400) in cells utilizing TRBV segments encoded within a 66 kb genomic region between TRBV23 and TRBV30. Our findings provide a comprehensive resource for anti-mouse TCR mAb specificity and insight into V-gene usage biases and T cell function.

Abstract Immune checkpoint inhibitor therapy leads to a strong and durable response in some metastatic melanoma patients, but some patients remain refractory to treatment. Identifying predictive biomarkers for therapeutic response is pivotal in advancing immunotherapy for melanoma. In this study, we analyzed 52 stage III and IV melanoma patients (14 non-responders and 38 responders) undergoing first-line immune checkpoint inhibitors therapy, of which 47 patients received anti-PD1 + anti-CTLA-4 and 5 patients received anti-PD-1 alone. Our main objective was uncovering factors associated with therapeutic response. Methods included multiplex bead array for plasma analytes, flow cytometry for immune profiling in blood and tumors, exome and transcriptome tumor sequencing, and scRNA-Seq for characterizing leukocytes in blood and tumor samples. Among 31 cytokines evaluated, CXCL9 emerged as a strong predictor of response, distinguishing responders prior to treatment and sustaining elevated levels throughout therapy in responders. Flow cytometry revealed responder blood samples enriched with CD8+ T lymphocytes expressing activation and cytotoxic markers, in addition to CXCR3, the receptor for CXCL9. Notably, TIM-3 correlated with complete versus partial response. On the other hand, non-responders showed elevated levels of CD161, PD-1, CD95, and PD-L2. Genomic characterization linked responder tumors with higher tumor mutational burden and lower tumor heterogeneity. Transcriptomic analysis demonstrated higher expression of CXCL9, CXCR3 and IFNG in responder tumors, mirroring systemic immune status. Lastly, scRNA-Seq data showed responder T cells differentiating into populations expressing GNLY, GZMA, GZMB, PRF1, IFNG, IFNGR1, and CXCR3. Conversely, non-responder T cells exhibited markers like TGFBI, ENTPD1, STAT6, ITGAE, IRF4, and KLRB1. Our findings suggest CXCL9 and CXCR3-related markers as potential predictive biomarkers and indicate distinctive immune pathways in responders, providing insight into the mechanisms underlying clinical outcomes in melanoma immunotherapy. Citation Format: Amanda B Figueiredo, Guilherme FB Evangelista, Milton JB Silva, Larissa M Kuil, Clara M Cavalcanti, Katia LP Morais, Juliana S Apostolico, Robert Balderas, Rodrigo P Lopes, Israel T Silva, Alexandre Defelicibus, Renan Valieris, James Turner, Nayane AL Galdino, Iasmim P Santos, Stephanie MI Ferreira, Ananda D Lopes, Clovis AL Pinto, Rubens Chojniak, Joao P Duprat-Neto, Kenneth J Gollob. Predictive biomarkers and mechanistic insights in stage III/IV melanoma patients undergoing first-line immune checkpoint inhibitors therapy [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B046.

Human immunodeficiency virus (HIV) persistence during antiretroviral therapy (ART) is associated with heightened plasma interleukin-10 (IL-10) levels and PD-1 expression. We hypothesized that IL-10 and PD-1 blockade would lead to control of viral rebound following analytical treatment interruption (ATI). Twenty-eight ART-treated, simian immunodeficiency virus (SIV)mac239-infected rhesus macaques (RMs) were treated with anti-IL-10, anti-IL-10 plus anti-PD-1 (combo) or vehicle. ART was interrupted 12 weeks after introduction of immunotherapy. Durable control of viral rebound was observed in nine out of ten combo-treated RMs for >24 weeks post-ATI. Induction of inflammatory cytokines, proliferation of effector CD8(+) T cells in lymph nodes and reduced expression of BCL-2 in CD4(+) T cells pre-ATI predicted control of viral rebound. Twenty-four weeks post-ATI, lower viral load was associated with higher frequencies of memory T cells expressing TCF-1 and of SIV-specific CD4(+) and CD8(+) T cells in blood and lymph nodes of combo-treated RMs. These results map a path to achieve long-lasting control of HIV and/or SIV following discontinuation of ART.

Abstract Background: The effectiveness of immunotherapy varies significantly among patients with advanced non-small cell lung cancer (NSCLC). This study investigates distinct subpopulations of CD4+T cells in the peripheral blood of stage IV NSCLC patients treated with anti-PD-1 combined with chemotherapy to identify immune mechanisms and systemic markers associated with treatment response or failure. Methods: We analyzed a cohort of 33 treatment-naive stage IV NSCLC patients, categorizing them as responders or non-responders based on clinical outcomes at 9 weeks and 6 months post-therapy initiation. Peripheral blood mononuclear cells (PBMCs) were collected before treatment, and high-dimensional flow cytometry was employed to profile immune cell subpopulations. Additionally, single-cell RNA sequencing (scRNAseq) data from both the same patients in our cohort and an independent cohort were analyzed to expand and validate the findings. Results: Non-responders exhibited elevated levels of suppressive phenotype CD4+ T lymphocytes expressing IL-10, CTLA-4, and other inhibitory markers like CD161 and CD95. Conversely, responders showed higher frequencies of activated CD4+CD69+ T lymphocytes, as well as increased percentages of TCF-1+ Effector Memory, Effector, and Central Memory CD4+ T cells. Responders also demonstrated higher levels of TCF7 and CXCR3 expression, markers associated with effective T cell activation and migration to the tumor microenvironment. Furthermore, single-cell RNA sequencing from the same cohort revealed differentially expressed genes in CD4+ T cells between responders and non-responders. Responders exhibited upregulation of cytotoxic genes such as NKG7 and GNLY and downregulation of genes like IL7R. Non-responders had higher levels of CTLA4 and KLRB1 expression, markers associated with immune suppression. ROC analysis showed that the frequencies of CD4+IL-10+ and CD4+TCF-1+ T cells could predict response with an AUC of 0.84 (p < 0.007). Finally, an in vitro dual blockade study with anti-PD-1/CTLA-4 using non-responder PBMC demonstrated that combining these two drugs significantly increased IFN-gamma and granzyme B production in CD4+ and CD8+ T cells. Conclusion: This study highlights the critical role of distinct CD4+ T cell subpopulations in predicting therapeutic outcomes in NSCLC patients undergoing anti-PD-1 therapy with chemotherapy. These findings provide valuable insights into the immune mechanisms driving response to immunotherapy and propose potential biomarkers for patient stratification. Citation Format: Amanda Figueiredo, Guilherme Evangelista, Clara Cavalcanti, Kátia Morais, Thaiany Goulart Souza-Silva, Robert Balderas, Rodrigo Pestana Lopes, Juan Santos e Silva, Jonathan Avila, Helder Nakaya, Nayane Galdino, Larissa Melo Kuil, Ananda Domingues Lopes, James Turner, Helano Freitas, Jefferson Luiz Gross, Walderez O Dutra, Valdemir Lima, Kenneth J Gollob. Distinct CD4+ T cell subpopulations as predictive biomarkers for anti-PD-1 and chemotherapy response in advanced NSCLC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr PR-01.

Abstract Background: Chimeric antigen receptor (CAR) T cell therapy is effective in treating B-cell malignancies, but factors influencing the persistence of functional CAR+ T cells, such as product composition, patients’ lymphodepletion, and immune reconstitution, are not well understood. To shed light on this issue, we conducted single-cell multi-omics analysis of transcriptional, clonal, and phenotypic profiles from pre- to 1-month post-infusion of CAR+ and CAR− T cells from patients who received a donor-derived 4-1BB CAR product targeting CD19. Methods: Longitudinal blood samples and infusion products were obtained from 10 individuals with large B cell lymphoma or B-cell acute lymphoblastic leukemia (CARTELL cohort in Sydney Australia1). CD45+ cells and enriched CAR+ T cells were single cell sorted for simultaneous scRNA-seq, 41 barcoded proteins (N=41) and T cell receptors, n= 115,000 cells across 21 samples. Machine learning methods were used to identify subsets of immune cells associated with clinically relevant parameters. Results: Following infusion, CAR+ and CAR− T cells showed similar differentiation profiles with clonally expanded populations across heterogeneous phenotypes, demonstrating clonal lineages and phenotypic plasticity. These findings were validated by performing the same analysis on two publicly available scRNA-seq data sets from two cohorts 2, 3 of paediatric (N=18) and adult (N=32) individual with large B cell lymphoma who received CAR19 T cells. Analysis of mass-cytometry data from our cohort and from 28 patients with B cell lymphoma treated with commercial CAR19 T cells (axi-cel)4, identified an association of CAR− and also CAR+ T cells, both expressing an 2B4− CD11b+ CD8+ phenotype, with complete remission or progressive disease at 3 and 6 months post-infusion. Finally, we report additional analysis on the immunogenomics of circulating CAR T cells in two patients who developed CAR T cell induced lymphoma and compared it with ascites and CAR-Lymphoma cells. Discussion: These results suggest that CAR+ and CAR− CD8+ T cells share a differentiation trajectory terminating in an NK-like phenotype that is associated with clinical outcome, and that non-CAR-derived signals may influence patients’ immune recovery and outcomes.References 1.Bishop, D.C. et al. Development of CAR T-cell lymphoma in 2 of 10 patients effectively treated with piggyBac-modified CD19 CAR T cells. Blood 138, 1504-1509 (2021).2.Haradhvala, N.J. et al. Distinct cellular dynamics associated with response to CAR-T therapy for refractory B cell lymphoma. Nat Med 28, 1848-1859 (2022).3.Wilson, T.L. et al. Common Trajectories of Highly Effective CD19-Specific CAR T Cells Identified by Endogenous T-cell Receptor Lineages. Cancer Discov 12, 2098-2119 (2022).4.Good, Z. et al. Post-infusion CAR T(Reg) cells identify patients resistant to CD19-CAR therapy. Nat Med 28, 1860-1871 (2022). Citation Format: Raymond Y. Louie, Jerome Samir, Curtis Cai, Helen McGuire, Tim Amos, James Fergsuson, Ira Deveson, Thiruni Adikari, Martina Bonomi, David Bishop, Christopher E. Mason, Robert Balderas, Marco Ruella, Mandeep Singh, David Gottlieb, Emily Blyth, Ken Micklethwaite, Fabio Luciani. Harnessing single cell multi-omics data to identify predictors of clinical outcome in CD19 CAR T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 882.

Supplementary Figure from Progenitor Hierarchy of Chronic Myelomonocytic Leukemia Identifies Inflammatory Monocytic-Biased Trajectory Linked to Worse Outcomes

Chimeric antigen receptor (CAR) T cell therapy is effective in treating B cell malignancies, but factors influencing the persistence of functional CAR + T cells, such as product composition, patients’ lymphodepletion, and immune reconstitution, are not well understood. To shed light on this issue, here we conduct a single-cell multi-omics analysis of transcriptional, clonal, and phenotypic profiles from pre- to 1-month post-infusion of CAR + and CAR − T cells from patients from a CARTELL study (ACTRN12617001579381) who received a donor-derived 4-1BB CAR product targeting CD19. Following infusion, CAR + T cells and CAR − T cells shows similar differentiation profiles with clonally expanded populations across heterogeneous phenotypes, demonstrating clonal lineages and phenotypic plasticity. We validate these findings in 31 patients with large B cell lymphoma treated with CD19 CAR T therapy. For these patients, we identify using longitudinal mass-cytometry data an association between NK-like subsets and clinical outcomes at 6 months with both CAR + and CAR − T cells. These results suggest that non-CAR-derived signals can provide information about patients’ immune recovery and be used as correlate of clinically relevant parameters.

Chimeric antigen receptor (CAR) T cells have demonstrable efficacy in treating B-cell malignancies. Factors such as product composition, lymphodepletion and immune reconstitution are known to influence CAR +T cell survival and persistence, however the determinants of CAR +T cells’ fate and differentiation and how the patients’ reconstituting immune system influence therapy outcomes remain poorly understood. We applied single cell multi-omics analysis of RNA, protein and clonal profiles of CAR +T and PBMC in patients receiving donor-derived products from a piggybac CAR19 clinical trial (N=8 patients). We used these data to reconstruct a differentiation trajectory, which explained the observed phenotype and fate of CAR +and CAR −T cells within the first 30 days of post-infusion. Following initial lympho-depletion, endogenous CAR −CD8 +, γδ T cells and CAR+ T cells clonally expanded, and differentiated across heterogenous phenotypes. They transitioned from a dominant resting or proliferating state into precursor of exhausted T cells, and finally into a terminal NK-like phenotype. The subset of terminal NK-like CAR +T cells was associated with increased serum concentrations of proinflammatory cytokines including IL-12 and IL-18. Validation on a published cohort (61 patients) showed that this subset was associated with clinical outcome at 6 months. Finally, we analysed in detail one patient that developed CAR T cell induced lymphoma and discussed the impact of early phase therapy in determining this unexpected outcome. These results demonstrate that CAR- and CAR+ T cells share a differentiation profile which may be determined by non-CAR derived signals and which may influence patients’ immune-recovery and outcome. National Health And Medical Research Funding (Australia) (APP1121643, APP1128416) BD Bioscience

With advances in next generation sequencing, researchers are able to enjoy the ease of sequencing tens of thousands of single cells in one experiment. However, the large cell loss associated poses great challenges for immune receptor profiling from rare clinical samples. To address these challenges, we previously developed Smart Immune Receptor Sequencing (SmartIR-Seq) to simultaneously profile T cell receptor (TCR) sequences and whole transcriptome in single cells more efficiently. However, to better understand T cell functions, it is essential to develop a method to link TCR sequence information to its cognate antigens in the same assay. Here, we describe a high-dimensional SmartIR-Seq technology, SmartIR-SeqHD, which allows us to multiplex cognate antigen specificities, paired TCR sequences, and whole transcriptome in single cells. In SmartIR-SeqHD, we in vitro transcribed and translated peptide encoding DNA oligos to generate a library of peptides, which were then loaded onto MHCs using a previously developed UV exchange method. By crosslinking streptavidin molecule with a universal oligo, we annealed a DNA oligo which serves as the DNA barcode for that antigen-specificity. Next, tetramer-stained cells were single-cell sorted into plates and lysed, enabling DNA barcodes to be amplified together with mRNAs, including TCR transcripts. We added cell barcodes during PCR amplification and included molecular identifier on the DNA barcodes. The addition of one more “dimension” to SmartIR-Seq using DNA-barcoded pMHC tetramers allows screening for large panels of antigens on primary T cells affordably and quickly, thus further improving the efficiencies and flexibility in the analysis of CD8+ T cells from rare clinical samples. Supported by grants from NIH R33 IMAT

Background: COVID-19 is a major public health concern and recent studies have shown that pregnant women are more likely to get severely ill from COVID-19 compared to not pregnant women. Actively infected women or convalescents at time of delivery were recruited.Methods: This prospective cohort was enrolled at UHCMC, CCHS, and MHS, in Cleveland, Ohio (March-October 2020). Collected samples were maternal blood, amniotic fluid (if C-session), umbilical cord segment, cord blood, amnion, chorion and neonatal throat swab and breastmilk. Viral load and cytokines/antibodies level were determined by RT-qPCR and MSD, respectively. In vitro validation in SARS-CoV-2 infected NHBE cells was performed. Data analysis was performed in R, GraphPad Prism 9, and Microsoft Excel.Findings: Plasma TGF-β2 levels were significantly associated with asymptomatic/mild disease and inversely correlated with levels of IP-10, IL-6 and IL-8. Pre-treatment of human tracheobronchial epithelial cells with TGF-β2 led to significant increase of IL-6(IFN- β2) that led to the control of SARS-CoV-2 viral loads. Additionally, TGF-β2 pre-treatment suppressed the release of cytokines part of the in vivo cytokine storm such as IP-10, IL1b and IL-8 post-infection. Altogether this data suggests that TGF-β2 plays a protective role in SARS-CoV-2 infection by improving epithelial cells intrinsic antiviral function and by suppressing the expression of the cytokines associated with heightened inflammation in severe cases.Interpretation: Our study shows that TGF- β2 is a cytokine that has a protective role during SARS-CoV-2 infection in pregnancy and is associated with lower viral loads and pro-inflammatory cytokines release in in vitro infection.Funding: NIH NCATS CTSA Program grant UL1TR002548 to Case Western Reserve University andsupport from the Clinical Research Center of University Hospitals Cleveland Medical CenterDeclaration of Interest: The authors declare no conflict of interest. Ethical Approval: This was a prospective cohort study (March-October 2020) approved by the institutional review board at University Hospitals Cleveland Medical Center (UHCMC), the Cleveland Clinic Healthcare System (CCHS) and the Metro Health System (MHS) (Approval number: 20200479).

Therapeutic interventions to eradicate latent HIV and restore immune function in ART-treated infection have yet to show efficacy. We applied an integrated -omic approach to identify a distinct group of individuals with poor CD4 T-cell reconstitution (Senescent Immunologic non-responders, “Senescent-INRs”) and the highest frequencies of CD4 T cells with inducible HIV. Contrary to the notion that immune activation drives HIV persistence and immune dysfunction, leukocytes of these subjects expressed genes that regulate cellular senescence. Here, increased frequencies of Tregs and TGF-β signaling cascade expression coincided with downregulation of cell metabolism/cycling in PD1-expressing central memory CD4 T cells and with frequencies of cells with inducible HIV. This profile, driven by a β-hydroxybutyrate rich metabolic milieu, resulted in TGF-β associated latency establishment/maintenance. Our findings identify cellular senescence as a novel mechanism of HIV persistence, that can be targeted by PD-1 or TGF-β specific interventions that have shown safety and efficacy in cancer.

Spleen is a key organ for immunologic surveillance, acting as a firewall for antigens and parasites that spread through the blood. However, how spleen leukocytes evolve across the developmental phase, and how they spatially organize and interact in vivo is still poorly understood. Using a novel combination of selected antibodies and fluorophores to image in vivo the spleen immune environment, we described for the first time the dynamics of immune development across postnatal period. We found that spleens from adults and infants had similar numbers and arrangement of lymphoid cells. In contrast, splenic immune environment in newborns is sharply different from adults in almost all parameters analysed. Using this in vivo approach, B cells were the most frequent subtype throughout the development. Also, we revealed how infections - using a model of malaria - can change the spleen immune profile in adults and infants, which could become the key to understanding different severity grades of infection. Our new imaging solutions can be extremely useful for different groups in all areas of biological investigation, paving a way for new intravital approaches and advances.

cells may effectively predict CHB patients that can safely discontinue NA treatment.

Abstract Myeloblast expansion is a hallmark of disease progression and comprises CD34+ hematopoietic stem and progenitor cells (HSPC). How this compartment evolves during disease progression in chronic myeloid neoplasms is unknown. Using single-cell RNA sequencing and high-parameter flow cytometry, we show that chronic myelomonocytic leukemia (CMML) CD34+ HSPC can be classified into three differentiation trajectories: monocytic, megakaryocyte-erythroid progenitor (MEP), and normal-like. Hallmarks of monocytic-biased trajectory were enrichment of CD120b+ inflammatory granulocyte–macrophage progenitor (GMP)-like cells, activated cytokine receptor signaling, phenotypic hematopoietic stem cell (HSC) depletion, and adverse outcomes. Cytokine receptor diversity was generally an adverse feature and elevated in CD120b+ GMPs. Hypomethylating agents decreased monocytic-biased cells in CMML patients. Given the enrichment of RAS pathway mutations in monocytic-biased cells, NRAS-competitive transplants and LPS-treated xenograft models recapitulated monocytic-biased CMML, suggesting that hematopoietic stress precipitates the monocytic-biased state. Deconvolution of HSPC compartments in other myeloid neoplasms and identifying therapeutic strategies to mitigate the monocytic-biased differentiation trajectory should be explored. Significance: Our findings establish that multiple differentiation states underlie CMML disease progression. These states are negatively augmented by inflammation and positively affected by hypomethylating agents. Furthermore, we identify HSC depletion and expansion of GMP-like cells with increased cytokine receptor diversity as a feature of myeloblast expansion in inflammatory chronic myeloid neoplasms. This article is highlighted in the In This Issue feature, p. 476