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CMV infection is a significant complication after solid organ transplantation. We used single cell TCR αβ sequencing to determine how memory inflation impacts clonality and diversity of the CMV-responsive CD8 and CD4 T cell repertoire in the first year after transplantation in human subjects. We observed CD8 T cell inflation but no changes in clonal diversity, indicating homeostatic stability in clones. In contrast, the CD4 repertoire was diverse and stable over time, with no evidence of CMV-responsive CD4 T cell expansion. We identified shared CDR3 TCR motifs among patients but no public CMV-specific TCRs. Temporal changes in clonality in response to transplantation and in the absence of detectable viral reactivation suggest changes in the repertoire immediately after transplantation followed by an expansion with stable clonal competition that may mediate protection.

CMV is a major infectious complication following solid organ transplantation. Reactivation of CMV leads to memory inflation, a process in which CD8 T cells expand over time. Memory inflation is associated with specific changes in T cell function, including increased oligoclonality, decreased cytokine production, and terminal differentiation. To address whether memory inflation during the first year after transplantation in human subjects alters T cell differentiation and function, we employed single-cell–matched TCRαβ and targeted gene expression sequencing. Expanded T cell clones exhibited a terminally differentiated, immunosenescent, and polyfunctional phenotype whereas rare clones were less differentiated. Clonal expansion occurring between pre- and 3 mo posttransplant was accompanied by enhancement of polyfunctionality. In contrast, polyfunctionality and differentiation state were largely maintained between 3 and 12 mo posttransplant. Highly expanded clones had a higher degree of polyfunctionality than rare clones. Thus, CMV-responsive CD8 T cells differentiated during the pre- to posttransplant period then maintained their differentiation state and functional capacity despite posttransplant clonal expansion.

Currently approved anti-influenza drugs target viral proteins, are subtype limited, and are challenged by rising antiviral resistance. To overcome these limitations, we sought to identify a conserved essential RNA secondary structure within the genomic RNA predicted to have greater constraints on mutation in response to therapeutics targeting this structure. Here, we identified and genetically validated an RNA stemloop structure we termed PSL2, which serves as a packaging signal for genome segment PB2 and is highly conserved across influenza A virus (IAV) isolates. RNA structural modeling rationalized known packaging-defective mutations and allowed for predictive mutagenesis tests. Disrupting and compensating mutations of PSL2’s structure give striking attenuation and restoration, respectively, of in vitro virus packaging and mortality in mice. Antisense Locked Nucleic Acid oligonucleotides (LNAs) designed against PSL2 dramatically inhibit IAV in vitro against viruses of different strains and subtypes, possess a high barrier to the development of antiviral resistance, and are equally effective against oseltamivir carboxylate-resistant virus. A single dose of LNA administered 3 days after, or 14 days before, a lethal IAV inoculum provides 100% survival. Moreover, such treatment led to the development of strong immunity to rechallenge with a ten-fold lethal inoculum. Together, these results have exciting implications for the development of a versatile novel class of antiviral therapeutics capable of prophylaxis, post-exposure treatment, and “just-in-time” universal vaccination against all IAV strains, including drug-resistant pandemics.One Sentence SummaryTargeting a newly identified conserved RNA structure in the packaging signal region of influenza segment PB2 abrogates virus production in vitro and dramatically attenuates disease in vivo.

Stem cell-based approaches have the potential to address the organ shortage in transplantation. Whereas both embryonic stem cells and induced pluripotent stem cells have been utilized as cellular sources for differentiation and lineage specification, their relative ability to be recognized by immune effector cells is unclear. We determined the expression of immune recognition molecules on hepatocyte-like cells (HLC) generated from murine embryonic stem cells and induced pluripotent stem cells, compared to adult hepatocytes, and we evaluated the impact on recognition by natural killer (NK) cells. We report that HLC lack MHC class I expression, and that embryonic stem cell-derived HLC have higher expression of the NK cell activating ligands Rae1, H60, and Mult1 than induced pluripotent stem cell-derived HLC and adult hepatocytes. Moreover, the lack of MHC class I renders embryonic stem cell-derived HLC, and induced pluripotent stem cell-derived HLC, susceptible to killing by syngeneic and allogeneic NK cells. Both embryonic stem cell-derived HLC, and induced pluripotent stem cell-derived HLC, are killed by NK cells at higher levels than adult hepatocytes. Finally, we demonstrate that the NK cell activation receptor, NKG2D, plays a key role in NK cell cytotoxicity of embryonic stem cell-derived HLC, but not induced pluripotent stem cell-derived HLC.

Immune responses generally decline with age. However, the dynamics of this process at the individual level have not been characterized, hindering quantification of an individual’s immune age. Here, we use multiple ‘omics’ technologies to capture population- and individual-level changes in the human immune system of 135 healthy adult individuals of different ages sampled longitudinally over a nine-year period. We observed high inter-individual variability in the rates of change of cellular frequencies that was dictated by their baseline values, allowing identification of steady-state levels toward which a cell subset converged and the ordered convergence of multiple cell subsets toward an older adult homeostasis. These data form a highdimensional trajectory of immune aging (IMM-AGE) that describes a person’s immune status better than chronological age. We show that the IMM-AGE score predicted all-cause mortality beyond well-established risk factors in the Framingham Heart Study, establishing its potential use in clinics for identification of patients at risk. REPORTING SUMMARY. Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.

Furosemide, a widely prescribed diuretic for edema-forming states, inhibits sodium reabsorption in the thick ascending limb of the nephron. Tubular adaptation to diuretics has been observed, but the range of mechanisms along the nephron has not been fully explored. Using morphometry, we show that furosemide induces renal tubular epithelial hyperplasia selectively in distal nephron segments. By comparison, we find progressive cellular hypertrophy in proximal and distal nephron segments. We next utilize single cell RNA sequencing of vehicle- and furosemide-treated mice to define potential mechanisms of diuretic resistance. Consistent with distal tubular cell hyperplasia, we detect a net increase in DCT cell number andBirc5, an anti-apoptotic and pro-growth gene, in a subset of DCT cells, as the most prominently up-regulated gene across the nephron. We also map a gradient of cell-specific transcriptional changes congruent with enhanced distal sodium transport. Furosemide stimulates expression of the mitogen IGF-1. Thus, we developed a mouse model of inducible deletion of renal tubular IGF-1 receptor and show reduced kidney growth and proximal, but not distal, tubular hypertrophy by furosemide. Moreover, genes that promote enhanced bioavailability of IGF-1 includingIgfbp1andIgfbp5are significantly and differentially expressed in proximal tubular segments and correspond to IGF-1R-dependent hypertrophy. In contrast, downstream PI3-kinase signaling genes includingPdk1, Akt1, Foxo3, FKBP4, Eif2BP4, andSpp1are significantly and differentially expressed in distal nephron segments and correspond to IGF-1R-independent hypertrophy. These findings highlight novel mechanisms of tubular remodeling and diuretic resistance, provide a repository of transcriptional responses to a common drug, and expand the implications of long-term loop diuretic use for human disease.

An early biomarker would transform our ability to screen and treat patients with cancer. The large amount of multi-scale molecular data in public repositories from various cancers provide unprecedented opportunities to find such a biomarker. However, despite identification of numerous molecular biomarkers using these public data, fewer than 1% have proven robust enough to translate into clinical practice. One of the most important factors affecting the successful translation to clinical practice is lack of real-world patient population heterogeneity in the discovery process. Almost all biomarker studies analyze only a single cohort of patients with the same cancer using a single modality. Recent studies in other diseases have demonstrated the advantage of leveraging biological and technical heterogeneity across multiple independent cohorts to identify robust disease biomarkers. Here we analyzed 17149 samples from patients with one of 23 cancers that were profiled using either DNA methylation, bulk and single-cell gene expression, or protein expression in tumor and serum. First, we analyzed DNA methylation profiles of 9855 samples across 23 cancers from The Cancer Genome Atlas (TCGA). We then examined the gene expression profile of the most significantly hypomethylated gene, KRT8, in 6781 samples from 57 independent microarray datasets from NCBI GEO. KRT8 was significantly over-expressed across cancers except colon cancer (summary effect size=1.05; p < 0.0001). Further, single-cell RNAseq analysis of 7447 single cells from lung tumors showed that genes that significantly correlated with KRT8 (p < 0.05) were involved in p53-related pathways. Immunohistochemistry in tumor biopsies from 294 patients with lung cancer showed that high protein expression of KRT8 is a prognostic marker of poor survival (HR = 1.73, p = 0.01). Finally, detectable KRT8 in serum as measured by ELISA distinguished patients with pancreatic cancer from healthy controls with an AUROC=0.94. In summary, our analysis demonstrates that KRT8 is (1) differentially expressed in several cancers across all molecular modalities and (2) may be useful as a biomarker to identify patients that should be further tested for cancer.

An early biomarker would transform our ability to screen and treat patients with cancer. The large amount of multi-scale molecular data in public repositories from various cancers provide unprecedented opportunities to find such a biomarker. However, despite identification of numerous molecular biomarkers using these public data, fewer than 1% have proven robust enough to translate into clinical practice1. One of the most important factors affecting the successful translation to clinical practice is lack of real-world patient population heterogeneity in the discovery process. Almost all biomarker studies analyze only a single cohort of patients with the same cancer using a single modality. Recent studies in other diseases have demonstrated the advantage of leveraging biological and technical heterogeneity across multiple independent cohorts to identify robust disease biomarkers. Here we analyzed 17149 samples from patients with one of 23 cancers that were profiled using either DNA methylation, bulk and single-cell gene expression, or protein expression in tumor and serum. First, we analyzed DNA methylation profiles of 9855 samples across 23 cancers from The Cancer Genome Atlas (TCGA). We then examined the gene expression profile of the most significantly hypomethylated gene, KRT8, in 6781 samples from 57 independent microarray datasets from NCBI GEO. KRT8 was significantly over-expressed across cancers except colon cancer (summary effect size=1.05; p < 0.0001). Further, single-cell RNAseq analysis of 7447 single cells from lung tumors showed that genes that significantly correlated with KRT8 (p < 0.05) were involved in p53-related pathways. Immunohistochemistry in tumor biopsies from 294 patients with lung cancer showed that high protein expression of KRT8 is a prognostic marker of poor survival (HR = 1.73, p = 0.01). Finally, detectable KRT8 in serum as measured by ELISA distinguished patients with pancreatic cancer from healthy controls with an AUROC=0.94. In summary, our analysis demonstrates that KRT8 is (1) differentially expressed in several cancers across all molecular modalities and (2) may be useful as a biomarker to identify patients that should be further tested for cancer.

Precision medicine approaches such as ex vivo drug sensitivity screening (DSS) are appealing to inform rational drug selection in myelodysplastic syndromes (MDSs) and acute myeloid leukemia, given their marked biologic heterogeneity. We evaluated a novel, fully automated ex vivo DSS platform that uses high-throughput flow cytometry in 54 patients with newly diagnosed or treatment-refractory myeloid neoplasms to evaluate sensitivity (blast cytotoxicity and differentiation) to 74 US Food and Drug Administration–approved or investigational drugs and 36 drug combinations. After piloting the platform in 33 patients, we conducted a prospective feasibility study enrolling 21 patients refractory to hypomethylating agents (HMAs) to determine whether this assay could be performed within a clinically actionable time frame and could accurately predict clinical responses in vivo. When assayed for cytotoxicity, ex vivo drug sensitivity patterns were heterogeneous, but they defined distinct patient clusters with differential sensitivity to HMAs, anthracyclines, histone deacetylase inhibitors, and kinase inhibitors (P < .001 among clusters) and demonstrated synergy between HMAs and venetoclax (P < .01 for combinations vs single agents). In our feasibility study, ex vivo DSS results were available at a median of 15 days after bone marrow biopsy, and they informed personalized therapy, which frequently included venetoclax combinations, kinase inhibitors, differentiative agents, and androgens. In 21 patients with available ex vivo and in vivo clinical response data, the DSS platform had a positive predictive value of 0.92, negative predictive value of 0.82, and overall accuracy of 0.85. These data demonstrate the utility of this approach for identifying potentially useful and often novel therapeutic drugs for patients with myeloid neoplasms refractory to standard therapies.

In silico quantification of cell proportions from mixed-cell transcriptomics data (deconvolution) requires a reference expression matrix, called basis matrix. We hypothesize that matrices created using only healthy samples from a single microarray platform would introduce biological and technical biases in deconvolution. We show presence of such biases in two existing matrices, IRIS and LM22, irrespective of deconvolution method. Here, we present immunoStates, a basis matrix built using 6160 samples with different disease states across 42 microarray platforms. We find that immunoStates significantly reduces biological and technical biases. Importantly, we find that different methods have virtually no or minimal effect once the basis matrix is chosen. We further show that cellular proportion estimates using immunoStates are consistently more correlated with measured proportions than IRIS and LM22, across all methods. Our results demonstrate the need and importance of incorporating biological and technical heterogeneity in a basis matrix for achieving consistently high accuracy., Cell type deconvolution from bulk expression data rely on a reference expression matrix. Here, the authors introduce a basis matrix built using data from both healthy and diseased samples profiled on 42 platforms, reducing biases introduced by single-platform matrices built using healthy samples.

Modifications of histone proteins are fundamental to the regulation of epigenetic phenotypes. Dysregulations of histone modifications have been linked to the pathogenesis of diverse human diseases. However, identifying differential histone modifications in patients with immune-mediated diseases has been challenging, in part due to the lack of a powerful analytic platform to study histone modifications in the complex human immune system. We recently developed a highly multiplexed platform, Epigenetic landscape profiling using cytometry by Time-Of-Flight (EpiTOF), to analyze the global levels of a broad array of histone modifications in single cells using mass cytometry. In this review, we summarize the development of EpiTOF and discuss its potential applications in biomedical research. We anticipate that this platform will provide new insights into the roles of epigenetic regulation in hematopoiesis, immune cell functions, and immune system aging, and reveal aberrant epigenetic patterns associated with immune-mediated diseases.

Chromatin undergoes extensive reprogramming during immune cell differentiation. Here we report the repression of controlled histone H3 amino terminus proteolytic cleavage (H3ΔN) during monocyte-to-macrophage development. This abundant histone mark in human peripheral blood monocytes is catalyzed by neutrophil serine proteases (NSPs) cathepsin G, neutrophil elastase and proteinase 3. NSPs are repressed as monocytes mature into macrophages. Integrative epigenomic analysis reveals widespread H3ΔN distribution across the genome in a monocytic cell line and primary monocytes, which becomes largely undetectable in fully differentiated macrophages. H3ΔN is enriched at permissive chromatin and actively transcribed genes. Simultaneous NSP depletion in monocytic cells results in H3ΔN loss and further increase in chromatin accessibility, which likely primes the chromatin for gene expression reprogramming. Importantly, H3ΔN is reduced in monocytes from patients with systemic juvenile idiopathic arthritis, an autoinflammatory disease with prominent macrophage involvement. Overall, we uncover an epigenetic mechanism that primes the chromatin to facilitate macrophage development. Chromatin undergoes extensive reprogramming during immune cell differentiation. Here Kuo and colleagues uncover an epigenetic mechanism that primes the chromatin to facilitate macrophage development.

INTRODUCTION: The combination of venetoclax (VEN) and a hypomethylating agent (HMA) such as decitabine (DEC) is frequently used in acute myeloid leukemia (AML) for patients unfit for standard chemotherapy. However, there is a subset of patients who do not clinically respond to such combination therapies. To identify VEN + HMA non-responders prior to treatment we developed an assay on our automated, high throughput (HTS) ex vivo drug sensitivity platform to predict how a given patient will clinically respond to DEC + VEN within an ongoing, open label, phase II clinical trial (NCT03404193) for patients with newly diagnosed elderly/unfit or relapsed/refractory (R/R) AML. To maximize the predictive power of our assay, blast and T cells of baseline samples were phenotyped, using multiparameter flow cytometry. Identifying non-responding patients before they are treated is important as it would enable them to avoid a treatment failure, unnecessary toxicity, and prioritize an alternative treatment. Importantly, this approach would define a patient population with a high unmet medical need, warranting the development of new therapeutics specifically for this population. METHODS: In NCT03404193, participants received DEC IV over 1 hour on Days 1-10 for induction and on Days 1-5 after achieving complete remission (CR) or complete remission with incomplete count recovery (CRi) during consolidation/maintenance. Participants received VEN PO daily on Days 1-28 of Cycle 1 and on Days 1-21 of subsequent cycles. VEN could be stopped on Day 21 if bone marrow analysis showed blast clearance or hypocellularity. Treatment cycles repeated every 4-6 weeks for up to 24 cycles in the absence of disease progression or unacceptable toxicity. Fig. 1A outlines the treatment schedule for NCT03404193. The primary objective is overall response rate (ORR). Peripheral blood (PB) specimens were obtained at baseline and 3±1 days after treatment begin in Cycle 1. Specimens were shipped on ice and processed within 1-4 days of the blood draws. Following red blood cell (RBC) lysis, nucleated cells were seeded in multi-well plates and treated with VEN in combination with DEC. Cells were cultured for a total of 7 days. Ex vivo blast reduction and frequencies of T cell subtypes were evaluated via multiparametric flow cytometry analysis and compared to clinical response. RESULTS: 7 days after initiation of the DEC + VEN ex vivo treatment of baseline samples, blast counts from Responders (Rs, N=2) were markedly reduced compared to those from Non-Responders (NRs, N=4) (Fig. 1B). Furthermore, baseline samples from Rs (N=9) exhibited higher levels of both CD4+ and CD8+ naïve T cells than from NRs (N=7) (Fig. 1C). To assess whether the kinetics of blast reduction in vivo correlates with increased or decreased levels of certain immune cell subsets we re-defined Rs and NRs based on their baseline-normalized blast counts 2 or 4 days after treatment begin in Cycle 1. Rapid Responders, enriched for subjects who experienced CRs or CRi's, were defined as subjects with blast levels ≤50% 2 days, or ≤33% 4 days, after treatment begin. Rapid Responders (N=6) exhibited higher levels of CD4+ T cells at the evaluation time point (2-4 days after treatment begin) than subjects with slower blast reductions or increases in their blast counts (N=4) (Fig. 1D). CONCLUSIONS: Baseline samples may predict clinical response to DEC + VEN in ex vivo drug treatment and immunophenotyping assays and may consequently serve to identify non-responders early, allowing them to switch to a potentially more effective treatment regimen before the lack of clinical response to DEC + VEN would become apparent. Rapid reduction of blasts in vivo coupled with increases in CD4 T cell levels may be a prognostic indicator of good clinical response. Limitations of our laboratory study include small sample sizes. Efforts are underway to validate the trends reported here. Figure 1 Figure 1. Disclosures Lacher: Notable Labs: Current Employment, Current holder of stock options in a privately-held company. Richardson: Notable Labs: Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Schaffert: Notable Labs: Consultancy, Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Kita: Notable Labs: Current Employment, Current holder of stock options in a privately-held company. Santaguida: Notable Labs: Consultancy, Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months, Patents & Royalties. Anderson: Notable Labs: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months. Roselli: Notable Labs: Current Employment, Current holder of stock options in a privately-held company. Anderson: Notable Labs: Current Employment, Current holder of stock options in a privately-held company. Paul: Notable Labs: Current Employment, Current holder of stock options in a privately-held company. Gu: Notable Labs: Current Employment, Current holder of stock options in a privately-held company. Vanderlaag: Notable Labs: Current Employment, Current holder of stock options in a privately-held company; Bristol-Myers Squibb: Ended employment in the past 24 months. DiNardo: ImmuneOnc: Honoraria, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Agios/Servier: Consultancy, Honoraria, Research Funding; Forma: Honoraria, Research Funding; Foghorn: Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Novartis: Honoraria; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. Konopleva: Agios: Other: grant support, Research Funding; Ablynx: Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Stemline Therapeutics: Research Funding; Rafael Pharmaceuticals: Other: grant support, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; KisoJi: Research Funding; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Ascentage: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; AstraZeneca: Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Sanofi: Other: grant support, Research Funding; Calithera: Other: grant support, Research Funding; Cellectis: Other: grant support; Forty Seven: Other: grant support, Research Funding.

Background Myelodysplastic syndromes (MDS) and MDS/MPNs are heterogeneous disorders with various combinations of mutations and cytogenetic abnormalities associated with distinct clinical phenotypes, prognosis, and implications for targeted therapies. We previously demonstrated that ex vivo drug sensitivity screening (DSS) identified subgroups of MDS and MDS/MPNs with differing patterns of sensitivity to various drug classes including hypomethylating agents (HMAs), kinase inhibitors, and other small molecules. In this study, we used hierarchical clustering to identify MDS and MDS/MPN genomic subgroups in a large single-center cohort. We then examined associations between these genomic subgroups and ex vivo sensitivity to various drug classes in a cohort of patients with ex vivo DSS. Methods Patients: We identified 294 patients with MDS or MDS/MPNs who had cytogenetics and HemeSTAMP NGS panel (164 genes) performed at Stanford between June 2018 and June 2021. A separate, partially overlapping cohort of 60 patients had ex vivo DSS as described below. Genomic clusters: We used a hierarchical Dirichlet Process (HDP), incorporating mutations and cytogenetics, to identify genomic subgroups. We included pathogenic and likely pathogenic variants with VAF >2% and excluded variants of unknown significance. Ex vivo DSS: Fresh bone marrow aspirates and peripheral blood specimens were RBC-lysed and resuspended in serum-free media with cytokines (Spinner et al, Blood Adv 2020;4(12):2768-78). Samples were plated in 384-well microtiter plates and screened against a collection of up to 74 drugs and 36 drug combinations in triplicate. Specimens were treated for 72 hours and assayed using flow cytometry to assess for blast viability. Statistical analysis: An HDP model was trained on the cohort of 294 patients. To tune the hyperparameters of the model, the log-likelihood of the test data was optimized using cross validation combined with Gaussian Process Bayesian optimization. Inference using the trained model was performed on 60 patients with ex vivo DSS producing a genomic component distribution for each patient. Jensen-Shannon distance was then computed between each pair of patients using their genomic component distributions. Patients were then clustered via agglomerative clustering (average linkage and using a maximum distance cutoff of 0.5) using this distance matrix. Ex vivo sensitivity to drug classes was then compared across clusters using ANOVA on drug sensitivity per drug class averaged over each patient. Results Patient characteristics: Among all 294 patients, the median age was 73 years, 78% had MDS, 16% had CMML, and 6% had other MDS/MPNs. 45% had >5% blasts and 53% had higher risk disease with IPSS-R >3.5. 94% had at least 1 mutation or cytogenetic abnormality with a median of 2 mutations (range 0-7). Among the 60 patients with ex vivo DSS, the median age was 77 years, 82% had MDS, 18% had CMML or other MDS/MPN, 55% had >5% blasts, and 67% had higher risk disease. Genomic subgroups and clusters: An HDP model trained on all 294 patients identified 16 genomic subgroups. Applying these genomic subgroups to the 60 patients with ex vivo DSS, we identified 12 genomic clusters, of which 6 clusters were most common: cluster 0 (enriched for RUNX1/BCOR mutations, N=6), cluster 1 (enriched for TET2/SRSF2/ASXL1, N=13), cluster 3 (enriched for DNMT3A, N=8), cluster 6 (enriched for KRAS/NRAS, N=5), cluster 7 (enriched for STAG2/ASXL1, N=6), and cluster 10 (enriched for TP53/complex cytogenetics, N=5). Associations between genomic clusters and drug sensitivity: Ex vivo drug sensitivity for 60 patients, organized by genomic cluster, is shown in Figure 1A. Ex vivo sensitivity to various drug classes is shown for the most common clusters in Figure 1B. Cluster 10 (enriched for TP53/complex cytogenetics) demonstrated greater ex vivo sensitivity to proteasome inhibitors (p=0.018). In Cluster 6 (enriched for NRAS/KRAS), there was a trend towards greater ex vivo resistance to HMAs and PARP inhibitors (p=0.1 for both comparisons). Conclusions Hierarchical clustering identified distinct genomic subgroups of MDS and MDS/MPNs, which displayed differing ex vivo sensitivity to various drug classes. While the small sample size limits our analysis, these associations between genotype and drug sensitivity phenotype are hypothesis generating and have potential implications for personalized therapy in MDS and MDS/MPNs. Figure 1 Figure 1. Disclosures Spinner: Notable Labs: Honoraria. Schaffert: Notable Labs: Consultancy, Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Santaguida: Notable Labs: Consultancy, Current holder of individual stocks in a privately-held company. Kita: Notable Labs: Current Employment, Current holder of stock options in a privately-held company. Aleshin: Notable Labs: Consultancy. Greenberg: Notable Labs: Research Funding.

Background Myelodysplastic syndromes (MDS) are heterogeneous myeloid neoplasms characterized by ineffective hematopoiesis, dysplasia, cytopenias, and a variable risk of progression to acute myeloid leukemia (AML). The biologic heterogeneity of MDS and related myeloid neoplasms relates to the genomic complexity of these disorders with different combinations of cytogenetic abnormalities and mutations associated with distinct clinical phenotypes. Ex vivo drug sensitivity screening (DSS) is a promising tool that may inform personalized therapy in MDS, particularly in patients refractory to standard therapies such as hypomethylating agents (HMAs). We report our updated experience using a fully automated ex vivo DSS platform in 64 patients with MDS and related myeloid neoplasms and identify correlations between clinical and genomic features and ex vivo drug sensitivity. Methods Patients: Patients were evaluated at the Stanford MDS Center between September 2016 and May 2020 and had a diagnosis of MDS, MDS/MPN, or secondary AML. Bone marrow (BM) aspirate and peripheral blood (PB) samples were procured for mutation testing (164-gene panel) and ex vivo DSS (Notable Labs, Foster City, CA). Ex vivo DSS: Fresh BM aspirate and PB specimens were RBC-lysed and resuspended in serum-free media with cytokines as previously described (Spinner et al, Blood Adv 2020;4(12):2768-78). Samples were plated in 384-well microtiter plates and screened against a collection of up to 74 drugs and 36 drug combinations in triplicate. Specimens were treated for 72 hours and assayed using high-throughput, multi-parametic flow cytometry, gating on the blast population (expressing CD34, CD33, and/or HLA-DR) to assess for blast viability. Patient clustering & statistical analysis: The Euclidean distance metric and Ward minimum variance method were used to identify patient clusters with distinct ex vivo drug sensitivity patterns. A 1-way repeated measures ANOVA was used to identify drug classes with variable sensitivity across clusters and to identify associations between clusters and clinical variables, including blast count, mutations and cytogenetic groups, IPSS-R risk group, and prior HMA exposure. A generalized estimation equation model was used to identify associations between mutations and ex vivo drug sensitivity. Results Ex vivo DSS was performed in 64 patients with myeloid neoplasms including 43 with MDS (67%), 11 with MDS/MPNs (17%), and 10 with secondary AML (16%). The median age was 75 years (range 23-90) and 78% were male. The majority of patients had higher risk disease with IPSS-R >3.5 (66%), excess blasts (58%), and adverse cytogenetics or mutations (53%) by IPSS-R or the ELN classification. Patients had a median of 2 pathogenic mutations (range 0-7), with the most frequent including TET2, ASXL1, DNMT3A, SF3B1, RUNX1, STAG2, SRSF2, NRAS, KRAS, BCOR, TP53, and EZH2. The majority of patients (64%) had prior HMA exposure. Ex vivo DSS defined three distinct patient clusters with differential sensitivity to numerous drug classes (Figure 1). Cluster 1 (N=13) demonstrated the greatest ex vivo sensitivity to HMAs, HMA/venetoclax combinations, cytotoxic agents, kinase inhibitors, mTOR inhibitors, HDAC inhibitors, and PARP inhibitors, while cluster 3 (N=19) demonstrated the greatest ex vivo resistance (p Correlating specific mutations with ex vivo drug sensitivity, STAG2 mutations were associated with greater ex vivo sensitivity to HMAs (p=0.002), HMA/venetoclax combinations (p=0.003), kinase inhibitors (p=0.002), and PARP inhibitors (p=0.003). TP53 mutations were associated with greater ex vivo sensitivity to proteasome inhibitors (p=0.0002). Conclusions Ex vivo DSS defined distinct patient clusters with differential sensitivity to numerous drug classes. Specific mutations, such as STAG2 and TP53, were associated with greater ex vivo sensitivity to specific drug classes. A larger sample size is needed to evaluate combinations of mutations and better define associations between genotype and drug sensitivity phenotype. Ultimately, combining both genomics and functional screening may further refine personalized therapy selection for patients with MDS and related myeloid neoplasms. Figure Disclosures Spinner: Notable Labs: Honoraria. Schaffert:Notable Labs: Current Employment. Aleshin:Notable Labs: Consultancy. Santaguida:Notable Labs: Current Employment. Tada:Notable Labs: Current Employment, Current equity holder in private company. Greenberg:BMS: Research Funding; Aprea: Research Funding; H3 Biotech: Research Funding; Notable Labs: Research Funding.

Background: Cytomegalovirus (CMV) infection is a major complication of organ transplantation. CMV establishes lifelong latency in infected individuals. Healthy people are protected from CMV reactivation by immune memory, but immunosuppression limits protection in transplant recipients. Despite antiviral prophylaxis strategies, long-term survival is impaired. CMV-responsive memory CD8 T cells expand during the first year after transplantation in the absence of detectable viremia. The observed expansion correlates with protection against CMV. The objective of this study was to determine whether these cells are protective, utilizing innovative single cell sequencing analyses to probe their functional characteristics. Methods: Mononuclear cells from peripheral blood of heart and kidney transplant recipients pre- and three months and one year post-transplant were stimulated with immunodominant CMV peptides. Single IFN gamma+ CMV-responsive T cells were index sorted into 96 well plates, and nested PCR, barcoding, and sequencing performed to analyze T cell receptor (TCR) and a panel of functional genes. Results: Expanded CMV-responsive T cells tend to express a limited number of dominant CMV-responsive TCR clones. Clonally expanded CMV-responsive T cells express multiple antiviral proteins. The TCR repertoire may change from pre- to post-transplant. Once established at three months, clonal dominance is maintained until at least one year. The number of antiviral proteins expressed per clonally expanded T cell increases between these time points. Conclusions: Protection from CMV post-transplant correlates with maintenance of TCR clonality and increased number of functions per cell. The stability of clonality post-transplant contrasts with the expansion of the total T cell population, and suggests that clonal expansion in the absence of disease recapitulates the pre-existing repertoire. The increased number of functions suggests that the expanded population may become more protective. The potential impact on clinical practice would be to provide further information on the relationship between the T cell response to CMV and long-term outcomes after transplantation.

Recently developed single-cell profiling technologies hold promise to provide new insights including analysis of population heterogeneity and linkage of antigen receptors with gene expression. These technologies produce complex data sets that require knowledge of bioinformatics for appropriate analysis. In this minireview, we discuss several single-cell immune profiling technologies for gene and protein expression, including cytometry by time-of-flight, RNA sequencing, and antigen receptor sequencing, as well as key considerations for analysis that apply to each. Because of the critical importance of data analysis for high parameter single cell analysis, we discuss essential factors in analysis of these data, including quality control, quantification, examples of methods for high dimensional analysis, immune repertoire analysis, and preparation of analysis pipelines. We provide examples of, and suggestions for, application of these innovative methods to transplantation research.

e19013 Background: Understanding the heterogeneity of AML is necessary for developing targeted drugs and diagnostics. A key measure of heterogeneity is the variance in response to treatments. Previously, we developed an ex vivo flow cytometry drug sensitivity assay (DSA) that predicted response to treatments in myelodysplastic syndrome. Unlike bulk cell viability measures of other drug sensitivity assays, our flow cytometry assay provides single cell resolution. The assay measures a drug’s effect on the viability or functional state of specific cell types. Here we present the development of this technology for AML, with additional measurements of DNA-Seq and RNA-Seq. Using the data from this assay, we aim to characterize the heterogeneity in AML drug sensitivity and the molecular mechanisms that drive it. Methods: As an initial feasibility analysis, we assayed 1 bone marrow and 3 peripheral blood AML patient samples. For the DSA, the samples were cultured with six AML standard of care (SOC) compounds across seven doses, in addition to two combinations. The cells were stained to detect multiple cell types including tumor blasts, and drug response was measured by flow cytometry. For the multi-omics, the cells were magnetically sorted to enrich for blasts and then assayed using a targeted 400 gene DNA-Seq panel and whole bulk transcriptome RNA-Seq. For comparison with BeatAML, Pearson correlations between gene expression and venetoclax sensitivity were investigated. Results: In our drug sensitivity assay, we measured dose response curves for the six SOC compounds, for each different cell type across each sample. The dose responses had cell type specific effects, including differences in drug response between CD11b+ blasts, CD11b- blasts, and other non-blast populations. Integrating with the DNA-Seq and RNA-Seq data, known associations between ex vivo drug response and gene expression were identified with additional cell type specificity. For example, BCL2A1 expression was negatively correlated with venetoclax sensitivity in CD11b- blasts but not in CD11b+ blasts. To further corroborate, among the top 1000 genes associated with venetoclax sensitivity in BeatAML, 93.7% had concordant directionality in effect. Conclusions: Here we describe the development of an integrated ex vivo drug sensitivity assay and multi-omics dataset. The data demonstrated that ex vivo responses to compounds differ between cell types, highlighting the importance of measuring drug response in specific cell types. In addition, we demonstrated that integrating these data will provide unique insights on molecular mechanisms that affect cell type specific drug response. As we continue to expand the number of patient samples evaluated with our multi-dimensional platform, this dataset will provide insights for novel drug target discovery, biomarker development, and, in the future, informing treatment decisions.

Post-translational modifications of histone proteins and exchanges of histone variants of chromatin are central to the regulation of nearly all DNA-templated biological processes. However, the degree and variability of chromatin modifications in specific human immune cells remain largely unknown. Here, we employ a highly multiplexed mass cytometry analysis to profile the global levels of a broad array of chromatin modifications in primary human immune cells at the single-cell level. Our data reveal markedly different cell-type- and hematopoietic-lineage-specific chromatin modification patterns. Differential analysis between younger and older adults shows that aging is associated with increased heterogeneity between individuals and elevated cell-to-cell variability in chromatin modifications. Analysis of a twin cohort unveils heritability of chromatin modifications and demonstrates that aging-related chromatin alterations are predominantly driven by non-heritable influences. Together, we present a powerful platform for chromatin and immunology research. Our discoveries highlight the profound impacts of aging on chromatin modifications.

Understanding the consequences of tuning TCR signaling on selection, peripheral T cell function, and tolerance in the context of native TCR repertoires may provide insight into the physiological control of tolerance. In this study, we show that genetic ablation of a natural tuner of TCR signaling, mir-181a-1/b-1, in double-positive thymocytes dampened TCR and Erk signaling and increased the threshold of positive selection. Whereas mir-181a-1/b-1 deletion in mice resulted in an increase in the intrinsic reactivity of naive T cells to self-antigens, it did not cause spontaneous autoimmunity. Loss of mir-181a-1/b-1 dampened the induction of experimental autoimmune encephalomyelitis and reduced basal TCR signaling in peripheral T cells and their migration from lymph nodes to pathogenic sites. Taken together, these results demonstrate that tolerance can be modulated by microRNA gene products through the control of opposing activities in T cell selection and peripheral T cell function.

In silico quantification of cell proportions from mixed-cell transcriptomics data (deconvolution) requires a reference expression matrix, called basis matrix. We hypothesized that matrices created using only healthy samples from a single microarray platform would introduce biological and technical biases in deconvolution. We show presence of such biases in two existing matrices, IRIS and LM22, irrespective of the deconvolution method used. Here, we present immunoStates, a basis matrix built using 6160 samples with different disease states across 42 microarray platforms. We found that immunoStates significantly reduced biological and technical biases. We further show that cellular proportion estimates using immunoStates are consistently more correlated with measured proportions than IRIS and LM22, across all methods. Importantly, we found that different methods have virtually no effect once the basis matrix is chosen. Our results demonstrate the need and importance of incorporating biological and technical heterogeneity in a basis matrix for achieving consistently high accuracy.

Influenza A virus’s (IAV’s) frequent genetic changes challenge vaccine strategies and engender resistance to current drugs. We sought to identify conserved and essential RNA secondary structures within IAV’s genome that are predicted to have greater constraints on mutation in response to therapeutic targeting. We identified and genetically validated an RNA structure (packaging stem–loop 2 (PSL2)) that mediates in vitro packaging and in vivo disease and is conserved across all known IAV isolates. A PSL2-targeting locked nucleic acid (LNA), administered 3 d after, or 14 d before, a lethal IAV inoculum provided 100% survival in mice, led to the development of strong immunity to rechallenge with a tenfold lethal inoculum, evaded attempts to select for resistance and retained full potency against neuraminidase inhibitor-resistant virus. Use of an analogous approach to target SARS-CoV-2, prophylactic administration of LNAs specific for highly conserved RNA structures in the viral genome, protected hamsters from efficient transmission of the SARS-CoV-2 USA_WA1/2020 variant. These findings highlight the potential applicability of this approach to any virus of interest via a process we term ‘programmable antivirals’, with implications for antiviral prophylaxis and post-exposure therapy.

The prokaryotic type II CRISPR/Cas9 system has been adapted to perform targeted genome editing in cells and model organisms. Here, we describe targeted gene deletion and replacement in human cells via the CRISPR/Cas9 system using two guide RNAs. The system effectively generated targeted deletions of varied length, regardless of the transcriptional status of the target gene. It is notable that targeted gene deletions generated via CRISPR/Cas9 and two guide RNAs resulted in the formation of correct junctions at high efficiency. Moreover, in the presence of a homology repair donor, the CRISPR/Cas9 system could guide precise gene replacement. Our results illustrate that the CRISPR/Cas9 system can be used to precisely and effectively generate targeted deletions or gene replacement in human cells, which will facilitate characterization of functional domains in protein-coding genes as well as noncoding regulatory sequences in animal genomes.

Single-cell flow cytometric techniques have been indispensable to improving our understanding of the phenotype and function of immune cell subsets that are important in both rejection and tolerance post-transplant. Mass cytometry, or Cytometry by Time-of-Flight (CyTOF), is a single cell–based platform that utilizes antibodies conjugated to rare heavy metal ions for analysis of cellular proteins by a time-of-flight mass spectrometer. This new technology allows for the evaluation of over 40 simultaneous cellular parameters in a single sample because the limitation of spectral overlap, seen in conventional flow cytometry, is eliminated. In this review we discuss the current state of mass cytometry, describe the advantages and disadvantages over multi-parameter flow cytometry, introduce novel methods of high-dimensional data analysis and visualization, and review some recent studies using mass cytometry to profile the immune system of healthy people, and transplant recipients.

Background Myelodysplastic syndrome (MDS) patients who are refractory to hypomethylating agents (HMAs) have a poor prognosis with median survival Methods Study design: We performed a prospective feasibility study in 21 patients with HMA-refractory MDS enrolled at Stanford University from April 2018 through March 2019. All patients had a baseline bone marrow (BM) biopsy with BM aspirate and peripheral blood (PB) samples sent for mutation testing (596-gene panel, Tempus, Chicago, IL) and ex vivo DSS (Notable Labs, Foster City, CA). Ex vivo DSS: BM aspirate and PB specimens were RBC-lysed and re-suspended in serum-free media with cytokines. Samples were plated in 384-well microtiter plates and screened against FDA-approved and investigational drugs (up to 76) and drug combinations in triplicate. Specimens were treated for 72 hours and assayed using high-throughput, multi-parametic flow cytometry for cytotoxicity and differentiation (Blood 2016;128:5206). In silico CBM: Genomic data were input into a computational biology model (Cell Works Group, San Jose, CA) to generate protein network maps for each patient. Mathematical modeling of MDS cell proliferation or inhibition was simulated for each patient and used to calculate drug efficacy scores for numerous agents (Leuk Res 2017;52:1-7). Study endpoints: Once the gene panel, ex vivo DSS, and in silico CBM results were available, we (M.A.S., A.A., J.Z., P.L.G.) met for a molecular tumor board (MTB) to review the data and provide personalized treatment recommendations for each patient. The primary endpoint was the feasibility of providing personalized recommendations within an actionable timeframe (≤30 days). Secondary endpoints included concordance between the ex vivo and in silico assays and the accuracy of our MTB recommendations in predicting clinical responses in vivo. Results The median age of the patients was 76 years (range 55-87) and 71% were male. Seventeen patients had MDS, 3 had an MDS/MPN disorder, and 1 patient had progressed to AML. 76% had higher risk disease by IPSS-R, 57% had excess blasts, and 52% had adverse cytogenetics or mutations. Patients had a median of 2 pathogenic mutations (range 0-6) with the most common including TET2, ASXL1, STAG2, DNMT3A, RUNX1, and SRSF2. The median turnaround time for results of the gene panel, ex vivo DSS, and in silico CBM were 14.5, 15, and 20 days, respectively. The median turnaround time to our MTB was 27 days (range 20-32 days). MTB recommendations varied widely among patients and encompassed various drug classes including targeted therapies (venetoclax, sorafenib, lenalidomide, ruxolitinib, midostaurin, everolimus), cytotoxic agents (cytarabine, fludarabine), differentiative agents (calcitriol, ATRA), HMAs, and androgens (danazol) as single agents or in combination. The ex vivo and in silico assays were highly concordant, particularly in predicting sensitivity to HMAs and venetoclax. Eight patients received treatment per our MTB recommendations. Of these 8 patients, 6 (75%) responded to the recommended therapy and 2 (25%) had stable disease. Two responding patients were bridged to allogeneic hematopoietic cell transplantation (HCT). The remaining patients elected for best supportive care (N=5), hospice (N=3), other approved therapies (N=3), a clinical trial (N=1), or allogeneic HCT without bridging therapy (N=1). Conclusions We demonstrate the feasibility of a novel precision medicine approach for HMA-refractory MDS patients combining mutation data, ex vivo DSS, and in silico CBM to guide clinical therapeutic decisions within an actionable timeframe. Personalized treatment recommendations accurately predicted clinical responses in vivo and enabled some patients to be bridged to allogeneic HCT. Randomized prospective trials are needed to determine whether this approach may improve outcomes for patients with HMA-refractory MDS. Disclosures Aleshin: Notable Labs: Consultancy. Santaguida:Notable Labs: Employment. Schaffert:Notable Labs: Employment. Abbasi:Cell Works Group, Inc.: Employment. Patterson:Notable Labs: Employment. Heiser:Notable Labs: Employment. Greenberg:Notable Labs: Research Funding; Celgene: Research Funding; Genentech: Research Funding; H3 Biotech: Research Funding; Aprea: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees.

Much has been learned about the molecular and cellular components critical for the control of immune responses and tolerance. It remains a challenge, however, to control the immune response and tolerance at the system level without causing significant toxicity to normal tissues. Recent studies suggest that microRNA (miRNA) genes, an abundant class of non-coding RNA genes that produce characteristic approximately 22 nucleotides small RNAs, play important roles in immune cells. In this article, we discuss emerging knowledge regarding the functions of miRNA genes in the immune system. We delve into the roles of miRNAs in regulating signaling strength and threshold, homeostasis, and the dynamics of the immune response and tolerance during normal and pathogenic immunological conditions. We also present observations based on analyzes of miR-181 family genes that indicate the potential functions of primary and/ or precursor miRNAs in target recognition and explore the impact of these findings on target identification. Finally, we illustrate that despite the subtle effects of miRNAs on gene expression, miRNAs have the potential to influence the outcomes of normal and pathogenic immune responses by controlling the quantitative and dynamic aspects of immune responses. Tuning miRNA functions in immune cells, through gain- and loss-of-function approaches in mice, may reveal novel approach to restore immune equilibrium from pathogenic conditions, such as autoimmune disease and leukemia, without significant toxicity.

Introduction: Myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML) and acute myeloid leukemia (AML) are clonal myeloid neoplasms for which limited conventional treatment options exist in the relapsed / refractory setting, especially for older patients. Methods: We provide proof of concept data by drug sensitivity profiling of 64 samples from 52 adult patients with both newly diagnosed and treatment refractory myeloid neoplasms (MDS = 38, CMML = 4, AML = 10). Fresh mononuclear cells from bone marrow aspirates and/or peripheral blood specimens were red blood cell lysed upon arrival and re-suspended in serum free media with cytokines. The samples were then plated in 384 well microtiter plates and were screened against a collection of investigational and FDA approved compounds (up to 85) in triplicate using a Labcyte Echo acoustic liquid handler. These specimens were treated for 72 hours and analyzed for drug sensitivity on an Intellicyt iQue Screener PLUS flow cytometer for both cytotoxicity and differentiation. Results: Principal component analysis was performed to explore differential ex vivo sensitivity and resistance patterns between the MDS and AML samples. Individual MDS samples clustered according to their ex vivo responses, with distinct subgroups enriched for sensitivity to hypomethylating agents (HMAs), HDAC inhibitors, differentiation agents, and PARP inhibitors. Strong clustering of drug classes was also observed with distinct drug classes showing high correlation within samples. Clinical parameters contributed but did not explain all of the variability of ex vivo sensitivity patterns, including prior HMA therapy, cytogenetics, prognostic risk category and mutational profile. In patients resistant to or relapsed from HMAs where next line of treatment matched profiled compounds (n = 19), ex vivo drug sensitivity data demonstrated positive (PPV) and negative predictive values (NPV) of 85% and 83%, respectively, for clinical response prediction (Fisher's exact test p-value = 0.0095). In patients with serial samples, ex vivo sensitivity data corresponded to emergence of clinical resistance. Conclusions: This novel platform, applied to predict ex vivo therapeutic responses of patient samples to various classes of drugs, recapitulates known clinical and molecular predictors of therapeutic efficacy and provides possible new biologically focused therapeutic options. The accuracy and reproducibility of this method coupled with short turnaround time demonstrate the potential of such an approach as a decision support system for therapeutic selection in the management of various myeloid neoplasms. A prospective study is ongoing to assess the feasibility of this technique for treatment decision support purposes in HMA-refractory MDS patients. Disclosures Aleshin: Mission Bio, Inc.: Consultancy; Natera, Inc.: Employment. Santaguida:Notable Labs: Employment. Patterson:Notable Labs: Employment. Gekas:Notable Labs: Employment. Schaffert:Notable Labs: Consultancy.

SUMMARY The expression of major histocompatibility complex class II (MHC II) molecules is post-translationally regulated by endocytic protein turnover. Here, we identified the serine protease cathepsin G (CatG) as an MHC II-degrading protease by in vitro screening and examined its role in MHC II turnover in vivo. CatG, uniquely among endocytic proteases tested, initiated cleavage of detergent-solubilized native and recombinant soluble MHC II molecules. CatG cleaved human leukocyte antigen (HLA)-DR isolated from both HLA-DM-expressing and DM-null cells. Even following CatG cleavage, peptide binding was retained by pre-loaded, soluble recombinant HLA-DR. MHC II cleavage occurred on the loop between fx1 and fx2 of the membrane-proximal beta2 domain. All allelic variants of HLA-DR tested and murine I-A(g7) class II molecules were susceptible, whereas murine I-E(k) and HLA-DM were not, consistent with their altered sequence at the P1' position of the CatG cleavage site. CatG effects were reduced on HLA-DR molecules with DRB mutations in the region implicated in interaction with HLA-DM. In contrast, addition of CatG to intact B-lymphoblastoid cell lines (B-LCLs) did not cause degradation of membrane-bound MHC II. Moreover, inhibition or genetic ablation of CatG in primary antigen-presenting cells did not cause accumulation of MHC II molecules. Thus, in vivo, the CatG cleavage site is sterically inaccessible or masked by associated molecules. A combination of intrinsic and context-dependent proteolytic resistance may allow peptide capture by MHC II molecules in harshly proteolytic endocytic compartments, as well as persistent antigen presentation in acute inflammatory settings with extracellular proteolysis.

The molecular basis of gammadelta T cell receptor (TCR) recognition is poorly understood. Here, we analyze the TCR sequences of a natural gammadelta T cell population specific for the major histocompatibility complex class Ib molecule T22. We find that T22 recognition correlates strongly with a somatically recombined TCRdelta complementarity-determining region 3 (CDR3) motif derived from germ line-encoded residues. Sequence diversity around these residues modulates TCR ligand-binding affinities, whereas V gene usage correlates mainly with tissue origin. These results show how an antigen-specific gammadelta TCR repertoire can be generated at a high frequency and suggest that gammadelta T cells recognize a limited number of antigens.

Human B cells are the primary targets of Epstein–Barr virus (EBV) infection. In most cases, EBV infection is asymptomatic because of a highly effective host immune response, but some individuals develop self-limiting infectious mononucleosis, while others develop EBV-associated lymphoid or epithelial malignancies. The viral and immune factors that determine the outcome of infection are not understood. The EBV life cycle includes a lytic phase, culminating in the production of new viral particles, and a latent phase, during which the virus remains largely silent for the lifetime of the host in memory B cells. Thus, in healthy individuals, there is a tightly orchestrated interplay between EBV and the host that allows the virus to persist. To promote viral persistence, EBV has evolved a variety of strategies to modulate the host immune response including inhibition of immune cell function, blunting of apoptotic pathways, and interfering with antigen processing and presentation pathways. In this article, we focus on mechanisms by which dysregulation of the host B cell and immune modulation by the virus can contribute to development of EBV+ B cell lymphomas.

Epstein-Barr virus (EBV) is a γ-herpesvirus that is linked to the development of posttransplant lymphoproliferative disorder (PTLD) in solid organ recipients. We previously demonstrated that EBV(+) B cell lymphoma cell lines isolated from patients with PTLD produce human IL-10 as an autocrine growth factor. However, little is known regarding IL-10 regulation in B cells. Here we show that EBV infection markedly alters the expression of host B cell microRNA, a class of small noncoding RNA that is an important regulator of transcriptional and posttranscriptional gene expression. Gene arrays reveal unique microRNA profiles in EBV(+) B cell lymphoma lines from patients with PTLD, compared to normal B cells or in vitro generated EBV(+) lymphoblastoid cell lines. We show that microRNA-194 expression is uniquely suppressed in EBV(+) B cell lines from PTLD patients and that the 3'untranslated region of IL-10 is targeted by microRNA-194. Overexpression of microRNA-194 attenuates IL-10 production and increases apoptosis of EBV(+) B cell lymphoma lines. Together, these data indicate that EBV co-opts the host B cell microRNA network and specifically suppresses microRNA-194 to override control of IL-10 expression. Thus, modulation of microRNA-194 may constitute a novel approach to inhibiting proliferation of EBV(+) B cell lymphomas in PTLD.

Oncogenes, which are essential for tumor initiation, development, and maintenance, are valuable targets for cancer therapy. However, it remains a challenge to effectively inhibit oncogene activity by targeting their downstream pathways without causing significant toxicity to normal tissues. Here we show that deletion of mir-181a-1/b-1 expression inhibits the development of Notch1 oncogene-induced T cell acute lymphoblastic leukemia (T-ALL). mir-181a-1/b-1 controls the strength and threshold of Notch activity in tumorigenesis in part by dampening multiple negative feedback regulators downstream of NOTCH and pre-T cell receptor (TCR) signaling pathways. Importantly, although Notch oncogenes utilize normal thymic progenitor cell genetic programs for tumor transformation, comparative analyses of mir-181a-1/b-1 function in normal thymocyte and tumor development demonstrate that mir-181a-1/b-1 can be specifically targeted to inhibit tumor development with little toxicity to normal development. Finally, we demonstrate that mir-181a-1/b-1, but not mir-181a-2b-2 and mir-181-c/d, controls the development of normal thymic T cells and leukemia cells. Together, these results illustrate that NOTCH oncogene activity in tumor development can be selectively inhibited by targeting the molecular networks controlled by mir-181a-1/b-1., Author Summary Oncogenes elicit driving signals required for tumor initiation, development, and maintenance and are valuable targets for cancer therapy. However, oncogenes often have essential functions in normal cellular physiology and produce intracellular proteins that are difficult to inhibit with small molecule drugs without causing significant toxicity to normal tissues. Thus, one of the challenges in cancer therapy is to identify downstream networks that can be targeted to specifically dampen the oncogenic signals in tumor cells without harming normal tissues. In this study we demonstrate that deletion of a microRNA (miRNA) gene, mir-181a-1/b-1, specifically inhibits the activity of the Notch oncogene in tumorigenesis without causing significant defects in normal development. Although earlier studies have elegantly shown the essential role of NOTCH and pre-TCR signals in NOTCH-induced tumorigenesis, neither NOTCH nor pre-TCR signals can be targeted effectively for treatment of T-ALL with available drugs due to their weak therapeutic effects and severe toxicities. Our findings illustrate that dissecting the downstream targets of miRNAs can reveal the molecular networks that can be targeted to control tumor transformation caused by oncogenes. More importantly, our results illustrate that comparative studies on the pathways utilized by normal cells and tumor cells may reveal novel insights into how tumorigenic pathways may be selectively inhibited with limited damage to normal tissues.

Emerging evidence suggests that microRNAs (miRNAs), an abundant class of ∼22-nucleotide small regulatory RNAs, play key roles in controlling the post-transcriptional genetic programs in stem and progenitor cells. Here we systematically examined miRNA expression profiles in various adult tissue-specific stem cells and their differentiated counterparts. These analyses revealed miRNA programs that are common or unique to blood, muscle, and neural stem cell populations and miRNA signatures that mark the transitions from self-renewing and quiescent stem cells to proliferative and differentiating progenitor cells. Moreover, we identified a stem/progenitor transition miRNA (SPT-miRNA) signature that predicts the effects of genetic perturbations, such as loss of PTEN and the Rb family, AML1-ETO9a expression, and MLL-AF10 transformation, on self-renewal and proliferation potentials of mutant stem/progenitor cells. We showed that some of the SPT-miRNAs control the self-renewal of embryonic stem cells and the reconstitution potential of hematopoietic stem cells (HSCs). Finally, we demonstrated that SPT-miRNAs coordinately regulate genes that are known to play roles in controlling HSC self-renewal, such as Hoxb6 and Hoxa4. Together, these analyses reveal the miRNA programs that may control key processes in normal and aberrant stem and progenitor cells, setting the foundations for dissecting post-transcriptional regulatory networks in stem cells.

Cytokine-induced killer (CIK) cells are T cell derived ex vivo expanded cells with both NK and T cell properties. They exhibit potent anti-tumor efficacy against various malignancies in preclinical models and have proven safe and effective in clinical studies. We combined CIK cell adoptive immunotherapy with IL-12 cytokine immunotherapy in an immunocompetent preclinical breast cancer model. Combining CIK cells with IL-12 increased anti-tumor efficacy in vivo compared to either therapy alone. Combination led to full tumor remission and long-term protection in 75% of animals. IL-12 treatment sharply increased the anti-tumor efficacy of short-term cultured CIK cells that exhibited no therapeutic effect alone. Bioluminescence imaging based in vitro cytotoxicity and in vivo homing assays revealed that short-term cultured CIK cells exhibit full cytotoxicity in vitro, but display different tumor homing properties than fully expanded CIK cells in vivo. Our data suggest that short-term cultured CIK cells can be “educated” in vivo, producing fully expanded CIK cells upon IL-12 administration with anti-tumor efficacy in a mouse model. Our findings demonstrate the potential to improve current CIK cell-based immunotherapy by increasing efficacy and shortening ex vivo expansion time. This holds promise for a highly efficacious cancer therapy utilizing synergistic effects of cytokine and cellular immunotherapy.

Students entering graduate study in immunology face a sharp transition into the autonomous world of laboratory research and a steep learning curve for the techniques particular to immunology. Our goal was to expose new graduate students to essential research skills to facilitate the transition into rotation laboratories and eventually a thesis lab. At Stanford University, over the past three years, we incorporated a weeklong intensive orientation (“Startup”) involving a series of guided, hands-on projects and workshops designed to introduce incoming graduate students to experimental methods in immunology research. Our curriculum posed questions in both mouse and human immunology, answered through pre-validated experiments including flow cytometry, quantitative PCR and fluorescence microscopy. For example, cytokines were measured by qPCR and intracellular staining of T cells. Data visualization and analysis were introduced through exposure to R, Flowjo and Fiji. The students attended lunches with senior graduate students, dinners with faculty, and tours of core facilities. Surveys of participants before and after Startup revealed improvements in experimental/technical confidence. Also, students reported that Startup built class cohesion and facilitated the selection of rotation labs. In summary, we provide a refined recipe for introducing students to immunology research through hands-on experimental modules that mitigate the pedagogical barriers of first-year graduate school.

Liver allografts are well tolerated and other solid organ allografts, when transplanted concurrently with livers, show improved graft outcomes. However, the mechanisms underlying “hepatic tolerance” have yet to be elucidated. Previous data show that hepatic dendritic cells (HDC) have diminished antigen presenting and immune stimulatory function compared with DC in lymphoid tissue. It has been hypothesized that immature plasmacytoid (p)DC are inherently tolerogenic with studies supporting this hypothesis. Our work confirms that HpDC prolong cardiac allograft survival (p

T cell sensitivity to antigens is regulated during development to ensure proper immunity and tolerance. We previously showed that miR-181a regulates the strength and threshold of T cell receptor signaling by targeting negative feedback molecules. Here we used miR-181 knockout mice to examine the role of miR-181 miRNAs in selection and T cell reactivity to antigens. We found that loss of mir-181ab1 caused an increase in the percent of positively selected CD8 single-positive (SP) cells in the thymi of female HY TCR transgenic mice. However, loss of miR-181 had no effect on the deletion CD8 SP cells in the thymi of male HY mice. Moreover, we showed that loss of mir-181ab1 resulted in two-fold more self-reactive T cells in the periphery after immunization with a self-peptide derived from myelin oligodendrocyte glycoprotein (MOG). Paradoxically, knockout mice exhibited a delayed disease onset in a MOG-dependent mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). In particular, the central nervous systems of knockout mice during disease had similar percentages of MOG-specific T cells, but an underrepresentation of TH17 and T H1-polarized CD4 T cells compared to the periphery. These findings demonstrate that mir-181ab1 also plays a role in modulating the differentiation and function of self-reactive T cells. Together, data from our study illustrate the diverse roles of mir-181ab1 in selection and autoimmunity.

Abstract 470 MiRNA-mediated gene regulation represents a fundamental layer of post-transcriptional control of gene expression with diverse functional roles in normal development and tumorigenesis. Whereas some studies have shown that over-expression of miRNA genes may contribute to cancer development and progression, it is yet to be rigorously tested by the loss-of-function genetic approaches whether miRNA genes are required for cancer development and maintenance in mice. Here we show that mir-181a1/b1 coordinates Notch and pre-TCR signals during normal thymocyte differentiation and plays an essential role in development and onset of T-cell acute lymphoblastic leukemia (T-ALL) induced by some Notch mutations. Using gain-of-function and loss-of-function approaches, we demonstrated that mir-181a1/b1 controls Notch and pre-TCR receptor signals during the early stages of T cell development in the thymus by repressing multiple negative regulators of both pathways, including Nrarp, PTPN-22, SHP2, DUSP5, and DUSP6. These results illustrate that a single miRNA can coordinate multiple signaling pathways by modulating the timing and strength of signaling at different stages. Intriguingly, synergistic signaling between Notch and pre-TCR pathways is necessary for the development of T-ALL, and miR-181 family miRNAs are aberrantly expressed in T-ALL patients. These observations raise the possibility that mir-181a1/b1 might contribute to the onset or maintenance of T-ALL by targeting similar pathways in tumor cells as it does in normal thymic progenitor cells. In support of this notion, we found that loss of mir-181a1/b1 significantly delayed the onset and development of T-ALL induced by intracellular domain of Notch1 (ICN1) and caused a 32% increase in the median survival time from 41 days to 54 days in T-ALL mice. Importantly, we noted that loss of mir-181a1/b1 more efficiently repressed the leukemogeneic potential of cells with lower levels of ICN1 expression, suggesting that mir-181a1/b1 may be more effective in inhibiting T-ALL development induced by a Notch mutant with weaker signal strength. Indeed, we demonstrated that loss of mir-181a1b1 essentially blocked T-ALL development induced by the weaker Notch mutant and dramatically decreased mortality from 60% to 10% in these T-ALL mice. Since human Notch mutations identified in T-ALL patients generally have weaker signaling strength and lower oncogenic potential than that of ICN1, our findings indicate that mir-181a1/b1 may play an essential role in development of normal thymic progenitors and Notch-induced T-ALL and may be targeted to treat T-ALL patients harboring Notch mutations. Disclosures: No relevant conflicts of interest to declare.