Immunology & Cell Biology

PU.1 is an ETS-domain transcription factor that has critical roles in many aspects of hematopoiesis and immune cell fate and function. In addition, aberrant PU.1 expression has been implicated in the development of acute myeloid leukemia (AML). Loss of PU.1 during adult murine hematopoiesis results in the expansion of immature granulocytes suggesting that PU.1 plays an important role in granulocyte maturation. To understand the molecular underpinnings of this process, we combined gene expression, transcription factor binding and 3D genome analyses with conditional deletion of PU.1 in vivo. We find that in contrast to normal granulocytes, PU.1-deficient cells possessed a transcriptome of immature granulocytes, in line with their cellular phenotype. Furthermore PU.1-deficient granulocytes display altered 3D genome architecture with a significant loss of interactivity in regions bound by PU.1 in control cells. Overall, this study implicates PU.1 as a key regulator of granulocyte maturation and lineage commitment through control of transcriptional programs and 3D chromatin architecture.

Autoimmune diseases, such as systemic lupus erythematosus (SLE), are underscored by dysregulated B cell function including the production of autoantibodies, skewed population ratios, and aberrant signaling. Given that the family of nuclear factor kappa B (NF-{kappa}B) transcription factors govern responses to stimuli, survival, differentiation, and so forth understanding the intricate regulatory network of NF-{kappa}B in B cell biology is paramount for unraveling treatments for B cell-linked autoimmune diseases. Here, we focus on a negative regulator of NF-{kappa}B signaling, A20 (TNFAIP3), that deactivates NF-{kappa}B transcription factor translocation through the ubiquitination and deubiquitination of target proteins. Haploinsufficiency in A20 results in an autoimmune phenotype and mutations to A20 have been associated with SLE, suggesting implications to B cell function. To investigate the role of A20 in NF-{kappa}B in human B cells, we generated a TNFAIP3 knockout (KO) Raji cell line. Cells were stimulated with either anti-IgM or Resiquimod (R848) to activate distinct NF-{kappa}B signaling pathways. Using qRT-PCR, western blotting, and flow cytometry, we assessed differences in gene expression, protein production, and NF-{kappa}B activation. We observed key limitations in using Epstein-Barr virus transformed B cell lines to model inducible NF-{kappa}B signaling.

CD4+FOXP3+ regulatory T (Treg) cells maintain self-tolerance, restrain immune responses during inflammatory stimuli, and promote tissue function and repair. Treg cell lineage identity, stability, and function depend on specific DNA methylation patterns maintained by the epigenetic regulator, UHRF1. Aging disrupts DNA methylation patterns necessary for Treg cell-mediated lung repair in a cell-autonomous manner. Nevertheless, whether maintenance DNA methylation is necessary for age-related Treg cell transcriptional and methylation programs is unknown. Here, we performed transcriptional and DNA methylation profiling on young and old Treg cells isolated from mice with chimeric Treg cell-specific loss of UHRF1. We observed cell-autonomous, age-related alterations in transcriptional and DNA methylation signatures that were dependent on UHRF1. We conclude that maintenance DNA methylation is required for age-related alterations in Treg cell transcriptional and DNA methylation signatures.

Conventional type 1 dendritic cells (cDC1) specialize in cross-presentation and interleukin-12 production and are critical for immunity against intracellular pathogens and tumors, but remain rare in vivo, limiting mechanistic and translational studies. Existing bone marrow-derived dendritic cell (BMDC) methods do not achieve highly selective enrichment of cDC1 or scalable production at high purity. Here, we established a novel in vitro culture system for selective generation of CD103+ cDC1 from mouse bone marrow using defined media conditions together with recombinant FLT3L, GM-CSF, and Kit ligand (KitL), termed iDC1. iDC1 cultures enabled scalable generation of an estimated 1.5 x 109 CD103+ cDC1 at greater than 95% purity from a single mouse, representing at least a 75-fold increase relative to previous recombinant cytokine-based methods. Phenotypic and transcriptional analyses demonstrated that iDC1 closely align with the CD103+ cDC1 lineage while remaining clearly distinct from macrophage populations. Functionally, iDC1 responded robustly to innate stimulation, produced interleukin-12 and inflammatory chemokines, and efficiently cross-presented cell-associated antigen to CD8+ T cells. Mechanistically, KitL and GM-CSF regulated distinct stages of cDC1 generation, whereas proteomic, phospho-proteomic, and functional analyses demonstrated that GM-CSF suppresses apoptosis and oxidative stress while promoting cDC1 proliferation. iDC1 generation was dependent on the +32 kb Irf8 enhancer required for bona fide cDC1 development, and STAT5-and BRD4-associated regulatory programs were identified as important regulators of efficient iDC1 generation. Together, these findings establish iDC1 cultures as a scalable platform for studying cDC1 biology and developing cDC1-based immunotherapeutic strategies.

Macrophage identity is widely viewed as a product of ontogeny and tissue-specific imprinting. Here, we identify a conserved, chromatin-driven maturation checkpoint that operates independently of initial lineage commitment and broadly across tissue contexts. Using long-term bone marrow-derived macrophage cultures, we uncover a late-stage transition characterized by coordinated transcriptional an epigenomic remodeling. Integration with in vivo developmental, tissue-resident, and monocyte repopulation datasets demonstrates that this program is conserved across ontogenies, tissues, species, and experimental systems, revealing a previously unrecognized stage of macrophage maturation. Functionally, late maturation preserves core macrophage activities while promoting lysosomal expansion and fundamentally rewiring innate immune responsiveness. Mature macrophages display enhanced stimulus-specific responses to interferons and microbial danger signals, coupled to increased metabolic and inflammatory competence while restricting interferon-induced transcriptional memory. Together, our findings identify late macrophage maturation as a conserved regulatory checkpoint that reprograms the logic of innate immune responsiveness through chromatin remodeling shaping innate immune function.

Numbers of ARID3a (AT-Rich Interaction Domain 3a) -expressing B lymphocytes from patients with systemic lupus erythematosus (SLE) are associated with increased disease activity. Normally, ARID3a-expressing circulating naive B cells are rare, but in SLE naive B cells dramatically increase ARID3a expression. We found that in vitro stimulation of B lymphocytes from healthy individuals with a cocktail of cytokines and agonists induced ARID3a in a subset of activated naive B cells and in IgD-CD27- double negative B cells previously associated with autoimmunity. Single cell RNA-seq of isolated naive B cells from ten SLE patients, with varying frequencies of ARID3a-expressing cells, revealed that ARID3a-associated genes included activation markers. Moreover, our data revealed the unexpected co-expression of the scavenger receptor CD68 with ARID3a, at both the transcript and protein level, in activated subsets of naive B cells. Inhibition of ARID3a in stimulated B cell cultures blocked naive B cell activation and CD68 expression. Together, these data identify ARID3a and CD68 as markers of naive B cell precursors associated with autoimmunity in SLE.

This dataset contains single-cell transcriptomic and surface protein profiles of bone marrow cells enriched for plasmacytoid dendritic cells (pDCs) and haematopoietic progenitor populations from septic and sham-operated mice. Sepsis was induced in BALB/c wild-type mice using cecal ligation and puncture (CLP) of moderate severity, with sham surgery as control. Bone marrow was collected 36 hours after surgery, depleted for selected lineage-positive cells by fluorescence-activated cell sorting, and processed using Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq). Sample demultiplexing was performed using hashtag oligonucleotides. The dataset includes gene expression matrices, antibody-derived tag counts, and barcode assignments for 13,725 cells, with a comparable distribution of sham and CLP samples across the total cell number. It may be used to study transcriptional states, surface marker expression, and phenotypic diversity of bone marrow cells in sepsis, to compare with other immunological and haematological single-cell datasets, or to train and benchmark computational tools for multimodal single-cell data analysis.

Bone marrow chimeras are widely used to study immune development and function. As the field moves from cesium-137 (137Cs)-based irradiators to X-ray irradiators for safety reasons, it is essential to determine if there are differences in immune system reconstitution after irradiating mice with one of these two radiation sources. Here, we performed a comprehensive immunological comparison of mice lethally irradiated with 137Cs or one of two different X-ray platforms and reconstituted with congenic bone marrow. Mice received 12 Gy total body radiation in two 6 Gy sessions followed by intravenous transfer of donor hematopoietic stem cells and were analyzed eight weeks post-transplant. We assessed mouse survival, donor chimerism, immune cell subset distribution, and activation states across multiple organs (bone marrow, spleen, lymph nodes, liver, and lung). All groups exhibited comparable survival and high levels of donor chimerism, with expected organ-specific reconstitution patterns. Immune lineage distributions, CD4/CD8 ratios, and activation states did not differ by irradiation type. Host-derived radioresistant cells were also similar across all irradiation groups and were predominantly composed of T cells skewed toward an activated phenotype. Overall, our data show that X-ray irradiation with proper filters and energy levels (225 KVp and 320 KVp) can yield equivalent immunological outcomes, including immune reconstitution and activation states, as compared to the same radiation dose from 137Cs-based irradiation in bone marrow chimera models. These results support the continued adoption of X-ray irradiation systems in place of 137Cs for generating bone marrow chimeras to be used across a wide range of immunologic studies.

BackgroundSevere eosinophilic asthma (SEA), eosinophilic granulomatosis with polyangiitis (EGPA) and nasal polyposis (NP) are immune-mediated diseases characterised by eosinophilic inflammation. However, there is also increasing interest in the potential pathological roles of autoantibodies in these diseases. Understanding their B cell receptor (BCR) repertoires may provide valuable insights into disease mechanisms, and potential role of B cells in their pathology. MethodsWe conducted BCR repertoire sequencing using peripheral blood from 43 patients, comprising SEA with nasal polyps (SEA+NP), SEA without nasal polyps (SEA-NP), and EGPA, along with 16 healthy controls (HCs). ResultsCompared to HCs, patients with EGPA exhibited increased relative proportions of IgA1, IgG1, IgG2, and IgG4 subclasses. Similarly, SEA-NP patients demonstrated significantly high proportion of IgG2 sequences. Notably, the IgG4 subclass was significantly elevated across all patient groups compared to HCs. Patients receiving anti-IL-5/5R biologic treatments showed increased relative proportions of IgA2 and IgG2 subclasses compared to untreated patients. Some variation across participant groups in mean somatic hypermutation and mutation frequency was evident. 1,508 clones shared across patients, but not healthy controls, were evident though the majority showed low clonal expansion. Nevertheless, a few shared clones did show either high prevalence across patients and/or higher clonal expansion. ConclusionChanges in BCR repertoires in SEA/EGPA are consistent with a pattern of a more mature B cell component in the periphery and with the T2 inflammatory response observed in SEA and EGPA. BCR clonotypes shared across patients were evident, however, whether such clonotypes are pathological in SEA/EGPA requires further investigation.

Abstract/SummaryWe present a streamlined protocol that enables the characterization of the metabolic state of immune cell populations through their distinct NADH/FAD autofluorescence fingerprints using a FACSymphony A5 spectral cytometer. We demonstrate the utility of this approach by profiling the metabolic status of diverse splenic B-cell subsets and assessing metabolic changes associated with their activation state.

RationaleEosinophilic airway inflammation is common in severe asthma and strongly associated with symptoms, exacerbations, and impaired lung function. Although type 2 (T2)-targeted biologics improve outcomes and reduce eosinophils, many patients experience residual symptoms and exacerbations. Emerging evidence suggests that these biologics may differentially affect specific airway eosinophil subpopulations, representing a potential mechanism of suboptimal treatment response. ObjectiveDetermine the effect of biologic treatment on eosinophil subpopulations in adults with severe asthma using in-depth immune profiling with mass cytometry (CyTOF). MethodsFifty adults with severe asthma (28 biologic-naive, 22 on stable-dose biologic therapy for [&ge;]6 months) underwent clinical phenotyping, spirometry, blood sampling, and sputum induction. Twenty-nine sputum samples passed quality control thresholds and were profiled by CyTOF. Manually gated sputum eosinophils were clustered using FlowSOM to identify eosinophil subpopulations, and cluster abundances and marker expression were compared across treatment groups. Measurements and Main ResultsCyTOF revealed treatment-associated shifts in circulating immune cells (lower CD4+ T cells and B cells, higher monocytes) and lower sputum CD8+ T cells. Unsupervised clustering of sputum eosinophils identified eight distinct subpopulations, and selective depletion of Cluster 6 was noted in biologic-treated participants (biologic-naive vs anti-TSLP logFC -4.98, p=0.003; biologic-naive vs anti-IL5 logFC -6.89, p=0.01). Higher Cluster 6 proportion correlated with worse ACT scores (rho = -0.44, p = 0.02) and lung function (FEV1 % predicted: rho = -0.47, p < 0.01; FEV1/FVC: rho = -0.40, p = 0.03). Functionally, Cluster 6 displayed enriched trafficking/activation markers (CCR3/Eotaxin-1, CD69, CD80, CRTH2) and non-T2 inflammatory mediators (TNF, IL-8, TLR7). ConclusionBiologic therapy in severe asthma was associated with selective depletion of a highly activated sputum eosinophil subpopulation with capability to drive both T2 and non-T2 inflammatory pathways. This cluster correlated with worse asthma control and lung function, indicating it may be a biologically important driver of persistent disease and potential biomarker to more accurately predict treatment response.

Infection by Mycobacterium tuberculosis (Mtb) is characterized by pathogen persistence in lung cells derived from blood monocytes. Since monocyte-derived lung subsets differ in their ability to restrict the growth of intracellular Mtb in mice, understanding the ontogeny of these subsets can inform development of host-directed therapies. Circulating monocytes are proposed to be heterogeneous, arising from distinct bone marrow or spleen progenitors that direct local differentiation. However, the role of the Mtb-infected lung environment in this process has not been addressed. We found that infected and uninfected mice had similar bone marrow monopoiesis, resulting in equivalent monocyte differentiation within the infected lung. While pulmonary Mtb infection also induced splenic monopoiesis, we found no impact on lung monocyte differentiation in splenectomized mice. However, when wildtype monocytes were transferred into Mtb-infected Sp140-/- recipients, in which excess Type I interferons and neutrophils alter the lung environment, we observed that donor-derived lung subsets resembled recipient-derived cells. In the lungs of Mtb-infected mice, we identified monocyte-derived lung subsets with unique gene expression, associated with specific spatial distributions and cell neighborhoods. These findings suggest that the local lung environment has a larger influence on the phenotypic diversity of monocyte-derived lung cells than does the peripheral environment.

{gamma}{delta} T cells are promising mediators of cancer immunotherapy, yet their potential to drive autoimmunity remains incompletely understood. Here, we identify a feedback mechanism in which innate-like V{gamma}1.1V{delta}6.3 T cells are reprogrammed into ILC1-like cells, thereby restraining autoimmune pathology. We define a previously unrecognized ILC1 subset whose development depends on an intact Tcrd locus. These cells predominantly harbor productive V{gamma}1.1 and V{delta}6 rearrangements, consistent with their origin from V{gamma}1.1V{delta}6.3 T cells. Mechanistically, TCR signaling induces Id3, which suppresses E protein-dependent activation of T cell-specific genes, including that encoding V{delta}6.3. Id3 ablation drives robust expansion of V{gamma}1.1V{delta}6.3 T cells and severe autoimmunity, characterized by tissue infiltration, autoantibody production, enhanced T follicular helper cell differentiation, and accumulation of germinal center and age-associated B cells. Together with previously described exocrine dysfunction, these features resemble human Sjogrens disease. Consistent with this, we observed in the salivary glands of Sjogrens disease patients an increased frequency of CD4-CD8- T cells enriched for {gamma}{delta} T cells, including subsets functionally analogous to murine V{gamma}1.1V{delta}6.3 cells. Collectively, these findings uncover a TCR-Id3-dependent reprogramming pathway that limit the pathogenic potential of harmful {gamma}{delta} T cells.

CD8 T cells play a central role in immune responses to infection and cancer. However, the diversity of T cell receptor (TCR) specificities makes it challenging to study the mechanisms that regulate T cell activation, differentiation, and effector function. Beyond TCR transgenic mouse models, various complex genome-editing approaches have been employed to overcome this challenge. However, these strategies are often technically demanding, time-intensive, and difficult to adapt. Investigators who are interested in testing de novo TCRs under their chosen experimental conditions would benefit from a standardized and accessible method. Here, we describe a protocol that combines ribonucleoprotein (RNP)-based CRISPR-Cas9 editing with retroviral transduction to enable efficient genetic manipulation of murine CD8 T cells. We show that T cells engineered via this protocol can be generated at sufficient scale for downstream in vitro assays and in vivo adoptive transfer experiments. We expect this method will be useful for investigators who require a standardized and accessible way to study how TCR specificity impacts CD8 T cell responses.

Major histocompatibility complexes (MHC) govern antigen presentation and T cell receptor (TCR) selection. Accurate in vivo modeling of human immunity therefore requires physiological human MHC TCR interactions. Humanized NOD scid IL2null (NSG) mice engrafted with human CD34+ hematopoietic stem cells are widely used to provide preclinical platforms for the development of advanced therapies; however, interactions between murine MHC and human TCR can promote xenoreactivity and alter T cell development. Here, we investigated how elimination of murine MHC together with different conditioning regimens shapes human T cell maturation in vivo. CD34+ cells from ten cord blood donors were transplanted into conventional NSG mice or murine MHC deficient NSG derivatives (DKO) following either sublethal irradiation or myeloablative busulfan conditioning. Integrated analyses combining flow cytometry, plasma cytokine profiling, and bulk and single cell TCR sequencing revealed marked differences in T cell differentiation across models. Busulfan conditioned DKO mice developed highly proliferative, activated, and cytotoxic T cells together with clonally expanded TCR repertoires. In contrast, irradiated NSG mice preferentially accumulated naive, NKT, and regulatory T cell populations. Busulfan-conditioned DKO mice showed no evidence of xenogeneic graft versus host disease and represent a refined enabling platform for human T cell development and provide a foundation for future preclinical evaluation of advanced gene and cell therapies.

Monocytes are critical regulators of allergic inflammation, whose functions are modified by IgE-driven processes. Monocytes are heterogeneous; comprised of multiple subsets which implies differential functions. In allergic inflammation, this heterogeneity is likely influenced by IgE-mediated effects. We sought to identify phenotypically distinct monocyte subsets related to allergic disease and then further delineate functional differences in cytokine release and antiviral responses. Using high dimensional spectral flow cytometry, we identified monocyte surface phenotypes directly related to surface levels of the high affinity IgE receptor (Fc{epsilon}RI) and surface-bound IgE. Fc{epsilon}RI+IgE+ monocytes, or FIMs, correlated with allergic disease and the level of atopy (i.e. serum IgE levels) of individual subjects. The FIM population also had differential surface expression of other molecules of monocyte maturation, which closely resembled a type 2 conventional dendritic cell (cDC2) phenotype. Functionally, FIMs had enhanced antiviral responses and IgE-driven IL-10 cytokine release. Finally, we showed that FIMs could be identified at higher levels in lung tissue from individuals with asthma. This study supports that atopic disease drives differential monocyte phenotypes, with the FIM population, specifically, as a more mature cell population closely related to dendritic cells with enhanced antiviral responses. The presence of monocytes in lung tissue during lethal asthma exacerbation further supports a role in regulating tissue inflammatory responses in allergic airway disease.

T cell exhaustion within the tumor microenvironment drives CD8+ T cells into a dysfunctional state characterized by progressive loss of proliferative capacity and effector functions, thereby limiting anti-tumor immunity and therapeutic efficacy. To investigate the biochemical alterations associated with exhaustion, an in vitro model of CD8+ T cell exhaustion was established through chronic antigenic stimulation of murine OT-1 CD8+ T cells. Phenotypic, functional, metabolic, and transcriptional characterization confirmed the acquisition of an exhausted state. Single-cell Raman spectroscopy was subsequently employed to generate biochemical signatures of activated, and exhausted CD8+ T cells. Principal component analysis (PCA) of the Raman spectral data revealed distinct separation of these two cell subsets, reflecting underlying biochemical differences associated with their functional states. Differential Raman spectral features corresponding to nucleic acids, carbohydrates, proteins, and lipids contributed significantly to this segregation, reflecting altered metabolic and biosynthetic states during exhaustion progression. Classification of the spectral data using machine-learning algorithms enabled accurate segregation of activated and exhausted T cells. Collectively, this study demonstrates that single-cell Raman spectroscopy can distinguish exhausted CD8+ T cells in a label-free and non-destructive manner, highlighting its potential as a platform for immune profiling and monitoring T cell dysfunction.

The complexity of stem cell differentiation programs remains incompletely understood across stem cell types, including for human bone marrow mesenchymal stromal/stem (BM-MSCs) cells, a heterogeneous cell population orchestrating bone formation and establishing a functional hematopoietic niche in the bone marrow. BM-MSCs form and repair bone through the evolutionarily conserved process of endochondral ossification (EO), initiated by deposition of a transient cartilage template subsequently remodeled into bone and bone marrow tissues. Despite their considerable potential for skeletal regeneration, the early molecular and cellular events underlying BM-MSCs commitment to endochondral ossification remain elusive. To overcome donor-dependent variability in chondrogenic potential that limits mechanistic studies, we here exploit OssiGel as a potent chondro-inductive extracellular matrix offering robust recapitulation of endochondral ossification by BM-MSCs. Through multi-omics profiling of OssiGel-primed BM-MSCs, we identify rapid chromatin remodeling at chondrogenic enhancer regions as a prerequisite for lineage commitment. The emergence of a chondro-progenitor population is detected as early as 3 days in vivo, and correlates with successful EO recapitulation. Mechanistically, we identify LINC02511 as a novel enhancer-associated element involved in the onset of EO. We confirm presence of LINC02511 in human skeletal atlases, and its CRISPR-mediated silencing was shown to significantly impair EO. By integrating tissue engineering with single cell multi-omics profiling, our study provides a framework for deciphering BM-MSCs fate decisions, highlighting the role of enhancers and non-coding elements as key determinants of early lineage specification. These findings advance our understanding of BM-MSCs biology and will prompt their translational exploitation in regenerative medicine.

Weight cycling (i.e. cycles of weight gain, loss, and regain) is a growing health concern that has been shown to worsen risk of diabetes beyond that of obesity. Dendritic cells play a causal role in obesity-associated inflammation and metabolic disease. However, whether dendritic cells are impacted by weight cycling is not known. Here, we aimed to test the hypothesis that antigen presentation increases in dendritic cells with weight cycling. C57Bl/6J male mice were put on nutrient-matched low-fat or high-fat diets to elicit lean, weight gain, weight loss, and weight cycled groups. Adipose tissue immune cell populations and proteins related to signal 1, 2, and 3 of antigen presentation were analyzed by single cell-RNA sequencing and flow cytometry. Total adipose tissue dendritic cells, conventional type I and II dendritic cells, and monocyte-derived dendritic cells all increased with weight loss. Regarding major histocompatibility complex (MHC) protein expression, non-classical MHCIb proteins (Qa1, Qa2 and CD1d) were also highest in the weight loss group. Dendritic cells expressing the costimulatory molecule CD86 and the cytokine TNF were highest in the weight loss group. Finally, we assessed effector immune cell populations in the adipose tissue to understand the functional role of antigen presentation. CD8+ T cells, natural killer (NK) T cells, and NK cells also increased consistently with weight loss, but showed evidence of exhaustion. In sum, weight loss, but not weight cycling expands dendritic cells, increases signals for non-classical antigen presentation, and expands CD8+ T cells, NKT cells, and NK cells in the adipose tissue.

AML is an aggressive blood cancer associated with poor clinical outcomes. Chemotherapy remains the standard of treatment, but unfortunately relapse is very common, highlighting the need for alternative therapies. T cell dysfunction and exhaustion are prominent in AML and may represent a barrier to effective immunotherapy yet remains poorly studied in AML. DNA methylation is a major driver of T cell exhaustion and inhibition of de novo methylation can block exhaustion and restore T cell function in chronic viral infections and other cancers but is understudied in AML. Here, we investigated the impact of azacytidine (Aza), an FDA-approved hypomethylating agent, on T cell exhaustion in AML. Using a spontaneous AML mouse model and samples from patients with AML, we found that Aza treatment modulates T cell function. In vivo Aza-treatment of AML-bearing mice decreased tumor burden and reshaped CD8+ T cell states, with increases in frequencies of memory subsets and decreases in regulatory T cells (Tregs). Functionally, Aza treatment overcame the impaired proliferation displayed by both CD4 and CD8+ T cells in our model. DNA methylation sequencing of T cells after Aza treatment revealed hypomethylation and increased expression of stem-like precursor gene TCF7 and E2F2, a regulator of cell cycle progression and proliferation. Similar changes in phenotypes were observed in cultures of AML patient samples treated with Aza. Collectively, we show that Aza remodels epigenetic and functional states in AML and has the potential to reverse T cell exhaustion, with enhanced memory and proliferation capacity. Our work generates a mechanistic framework that provides rationale of combining hypomethylating agents with T cell-based immunotherapies in this lethal disease. Data Sharing StatementRRBS data is available in GEO under the accession number GSE328721. For original data please contact Dr. Evan F. Lind. Key PointsAzacytidine mediated epigenetic modulation can alleviate T cell exhaustion in AML Translational RelevanceImmune therapy has shown limited efficacy in AML, despite increasing evidence of T cell dysfunction in this malignancy. Azacytidine (Aza) is an FDA approved drug for AML, but patients develop therapy resistance and relapse. Studies have mainly focused on Azas tumor intrinsic effects. In this study, we investigated the impact of Aza on immune function, especially T cell exhaustion in AML, since exhaustion is a major mechanism of disease resistance. We demonstrated that Aza can modulate T cell phenotype and restore T cell proliferation. Mechanistically, Aza induces epigenetic reprogramming in T cells and increases the expression of a stem-like precursor marker, TCF7. By shifting the focus on T cell biology, our study provides a rationale for combining Aza with other immunotherapies that can enhance durable immune responses in this malignancy.