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Immune checkpoint inhibitor (ICI) therapies that promote T cell activation have improved outcomes for advanced malignancies yet also elicit harmful autoimmune reactions. The T cell mechanisms mediating these iatrogenic autoimmune events remain unclear. Here we assayed T cells from joints of patients affected by ICI-induced inflammatory arthritis (ICI-arthritis), which can present clinically indistinguishable from rheumatoid arthritis (RA). Compared to the autoimmune arthritides RA and psoriatic arthritis (PsA), ICI-arthritis joints contained an expanded CD38hi CD127− CD8+ T cell subset that displays cytotoxic, effector, and interferon (IFN) response signatures. The abundance of CD38hi CD8 T cells in ICI-arthritis resulted from a limited number of clones that could be found proliferating in the joint. Exposure of synovial T cells to Type I IFN, more so than IFN-γ, induces the CD38hi cytotoxic phenotype. Relative to other CD8+ T cell subsets in the joints, the CD38hi population is distinct from a dysfunctional population and clonally most related to TCF7+ memory populations. Examination of synovial tissue from bilateral knee arthroplasty demonstrated considerable sharing of TCR clonotypes in the CD38hi CD8 T cell fraction from both knees. These results define a distinct CD8 T cell subset that may be directly activated by ICI therapy and mediate a tissue-specific autoimmune cellular reaction in patient joints.

Background Immunosuppressive and anti-cytokine treatment may have a protective effect for patients with COVID-19. Understanding the immune cell states shared between COVID-19 and other inflammatory diseases with established therapies may help nominate immunomodulatory therapies. Methods To identify cellular phenotypes that may be shared across tissues affected by disparate inflammatory diseases, we developed a meta-analysis and integration pipeline that models and removes the effects of technology, tissue of origin, and donor that confound cell-type identification. Using this approach, we integrated > 300,000 single-cell transcriptomic profiles from COVID-19-affected lungs and tissues from healthy subjects and patients with five inflammatory diseases: rheumatoid arthritis (RA), Crohn’s disease (CD), ulcerative colitis (UC), systemic lupus erythematosus (SLE), and interstitial lung disease. We tested the association of shared immune states with severe/inflamed status compared to healthy control using mixed-effects modeling. To define environmental factors within these tissues that shape shared macrophage phenotypes, we stimulated human blood-derived macrophages with defined combinations of inflammatory factors, emphasizing in particular antiviral interferons IFN-beta (IFN-β) and IFN-gamma (IFN-γ), and pro-inflammatory cytokines such as TNF. Results We built an immune cell reference consisting of > 300,000 single-cell profiles from 125 healthy or disease-affected donors from COVID-19 and five inflammatory diseases. We observed a CXCL10+ CCL2+ inflammatory macrophage state that is shared and strikingly abundant in severe COVID-19 bronchoalveolar lavage samples, inflamed RA synovium, inflamed CD ileum, and UC colon. These cells exhibited a distinct arrangement of pro-inflammatory and interferon response genes, including elevated levels of CXCL10, CXCL9, CCL2, CCL3, GBP1, STAT1, and IL1B. Further, we found this macrophage phenotype is induced upon co-stimulation by IFN-γ and TNF-α. Conclusions Our integrative analysis identified immune cell states shared across inflamed tissues affected by inflammatory diseases and COVID-19. Our study supports a key role for IFN-γ together with TNF-α in driving an abundant inflammatory macrophage phenotype in severe COVID-19-affected lungs, as well as inflamed RA synovium, CD ileum, and UC colon, which may be targeted by existing immunomodulatory therapies.

Additional file 2: Figure S1. Overall integration of immune cells from multiple scRNA-seq datasets. Figure S2. Quantification of the performance of all cell type multi-disease tissue integration. Figure S3. Tissue-level macrophage integrative analysis of multiple scRNA-seq datasets. Figure S4. Heterogeneity of shared inflammatory macrophages from multiple tissues. Figure S5. Single-cell differential gene expression analysis of comparing inflammatory macrophages with non-inflammatory macrophages within each individual tissue source. Figure S6. Examination of the CXCL10+ CCL2+ macrophage marker genes in additional diseased cohort studies. Figure S7. Experimental design and quality control of human blood-derived macrophages stimulated by different conditions. Figure S8. Integrative analysis of tissue-level macrophages and human blood-derived macrophages. Figure S9. Assessment of previously reported stimulated macrophage spectrum analysis and alignment of macrophages from different disease tissues to a trajectory.

Background Immunosuppressive and anti-cytokine treatment may have a protective effect for patients with COVID-19. Understanding the immune cell states shared between COVID-19 and other inflammatory diseases with established therapies may help nominate immunomodulatory therapies. Methods To identify cellular phenotypes that may be shared across tissues affected by disparate inflammatory diseases, we developed a meta-analysis and integration pipeline that models and removes the effects of technology, tissue of origin, and donor that confound cell-type identification. Using this approach, we integrated > 300,000 single-cell transcriptomic profiles from COVID-19-affected lungs and tissues from healthy subjects and patients with five inflammatory diseases: rheumatoid arthritis (RA), Crohn’s disease (CD), ulcerative colitis (UC), systemic lupus erythematosus (SLE), and interstitial lung disease. We tested the association of shared immune states with severe/inflamed status compared to healthy control using mixed-effects modeling. To define environmental factors within these tissues that shape shared macrophage phenotypes, we stimulated human blood-derived macrophages with defined combinations of inflammatory factors, emphasizing in particular antiviral interferons IFN-beta (IFN-β) and IFN-gamma (IFN-γ), and pro-inflammatory cytokines such as TNF. Results We built an immune cell reference consisting of > 300,000 single-cell profiles from 125 healthy or disease-affected donors from COVID-19 and five inflammatory diseases. We observed a CXCL10+ CCL2+ inflammatory macrophage state that is shared and strikingly abundant in severe COVID-19 bronchoalveolar lavage samples, inflamed RA synovium, inflamed CD ileum, and UC colon. These cells exhibited a distinct arrangement of pro-inflammatory and interferon response genes, including elevated levels of CXCL10, CXCL9, CCL2, CCL3, GBP1, STAT1, and IL1B. Further, we found this macrophage phenotype is induced upon co-stimulation by IFN-γ and TNF-α. Conclusions Our integrative analysis identified immune cell states shared across inflamed tissues affected by inflammatory diseases and COVID-19. Our study supports a key role for IFN-γ together with TNF-α in driving an abundant inflammatory macrophage phenotype in severe COVID-19-affected lungs, as well as inflamed RA synovium, CD ileum, and UC colon, which may be targeted by existing immunomodulatory therapies.

Immunosuppressive and anti-cytokine treatment may have a protective effect for patients with COVID-19. Understanding the immune cell states shared between COVID-19 and other inflammatory diseases with established therapies may help nominate immunomodulatory therapies. Using an integrative strategy, we built a reference by meta-analyzing > 300,000 immune cells from COVID-19 and 5 inflammatory diseases including rheumatoid arthritis (RA), Crohn’s disease (CD), ulcerative colitis (UC), lupus, and interstitial lung disease. Our cross-disease analysis revealed that an FCN1+ inflammatory macrophage state is common to COVID-19 bronchoalveolar lavage samples, RA synovium, CD ileum, and UC colon. We also observed that a CXCL10+ CCL2+ inflammatory macrophage state is abundant in severe COVID-19, inflamed CD and RA, and expresses inflammatory genes such as GBP1, STAT1, and IL1B. We found that the CXCL10+ CCL2+ macrophages are transcriptionally similar to blood-derived macrophages stimulated with TNF-α and IFN-γ ex vivo. Our findings suggest that IFN-γ, alongside TNF-α, might be a key driver of this abundant inflammatory macrophage phenotype in severe COVID-19 and other inflammatory diseases, which may be targeted by existing immunomodulatory therapies.

In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single-cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicit distinct differentiation and proliferation effects in single highly purified LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single-cell resolution., Previous gene editing in haematopoietic stem cells (HSCs) has focussed on a heterogeneous CD34+ population. Here, the authors demonstrate high efficiency CRISPR/Cas9-based editing of purified long-term HSCs using non-homologous end joining and homology-directed repair, by directing isoform-specific expression of GATA1.

Leukemia is the most common cancer in children. Sequencing data from identical twins suggests that the first genetic alterations in childhood leukemia occur in utero. Children with Down syndrome (Trisomy 21, T21) have an increased risk of childhood leukemia. In 30% of newborns with Down syndrome, a transient pre-leukemia disease occurs, which is characterized by a clonal proliferation of immature megakaryocytes carrying somatic mutations in the GATA1 transcription factor. These acquired GATA1 mutations lead to the expression of an N-terminal truncated protein (GATA1-Short). In 20% of the cases, acute megakaryoblastic leukemia (AMKL) evolves from the pre-leukemia by acquisition of additional genetic mutations in the transient leukemia clone, predominantly in genes of the cohesin complex. It is hypothesized that this represents a multi-step process of leukemogenesis with three distinct genetic events: T21, GATA1-Short and additional cohesin mutations. Yet, it remains unclear how an extra copy of chromosome 21 predisposes towards leukemia, the mechanisms of leukemic transformation and the interplay between each genetic component. Therefore, we wanted to establish a tractable human model system to investigate the initiation and evolution of transient leukemia and AMKL using CRISPR/Cas9 genome editing in primary human hematopoietic stem cells (HSCs). To model the initiation of Down syndrome associated pre-leukemia, we utilized both neonatal cord blood and fetal liver derived LT-HSCs and other progenitor populations to express either the short or long isoform of GATA1 (GATA1-Short or GATA1-Long). This was carried out using an improved methodology that permits the in vitro and in vivo functional interrogation of CRISPR/Cas9 edited human LT-HSCs at the single cell level (Wagenblast et al., bioRxiv 609040). Importantly, in this case, expression of either GATA1 isoform remained under the regulatory control of the endogenous promoter. Culture of single LT-HSC, short-term (ST-HSC) and myelo-erythroid progenitors (MEP) revealed a drastic shift towards megakaryocytic lineage output upon exclusive expression of GATA1-Short compared to control or GATA1-Long, regardless of the developmental source of the derived cells. To investigate the functional consequences of exclusive GATA1-Short expression in LT-HSCs in vivo, we performed near-clonal xenotransplantation assays in NSG and NSGW41 mice. Strikingly, GATA1-Short edited LT-HSCs injected mice displayed a higher percentage of human CD41+CD45- megakaryocytic lineage derived cells and a decrease in human GlyA+CD45- erythroid cells compared to control. Morphological analysis revealed more immature forms of erythroid cells and fewer enucleated erythrocytes in GATA1-Short edited LT-HSCs injected mice. In order to add an additional genetic determinant to our model, we utilized T21 fetal liver derived LT-HSCs. Un-manipulated T21 LT-HSCs and other progenitor populations showed a bias towards erythroid, myeloid and megakaryocytic lineages at the expense of lymphoid fates. In vitro, the combination of T21 and CRISPR/Cas9-mediated GATA1-Short in LT-HSCs led to an increase in megakaryocytic lineage output, while decreasing erythroid output. This phenotype was similar to what was observed in normal karyotype fetal liver derived LT-HSCs. However, near clonal transplantation of GATA1-Short edited T21 LT-HSCs in NSG mice generated exclusive CD33+ myeloid grafts with disproportionate high levels of CD41+CD45- megakaryocytic lineage derived cells compared to T21 control. In addition a distinct CD34+CD41+CD71+CD45+ population was present. Thus, this phenotype is reminiscent of Down Syndrome associated transient leukemia. In summary, by using an improved CRISPR/Cas9 single cell methodology we show how GATA1 regulates lineage fate in normal and T21 LT-HSCs and other progenitor populations. Importantly, we show for the first time a humanized mouse model of Down syndrome associated transient leukemia, which was induced from T21 human fetal liver derived LT-HSCs engineered to express GATA1-Short. Current studies focus on adding additional mutations of the cohesin complex to progress transient leukemia to AMKL. Disclosures No relevant conflicts of interest to declare.

Children with Down syndrome, also known as trisomy 21, have a significantly increased risk of childhood acute leukemia in the first few years after birth. The acute leukemia phase is preceded with a transient pre-leukemia phase in newborns, which is characterized by a clonal proliferation of immature megakaryocytes carrying somatic mutations in the GATA binding protein 1 (GATA1). These acquired GATA1 mutations lead to the expression of the GATA1 short isoform and prevent the expression of the GATA1 long isoform. The pre-leukemia undergoes spontaneous remission within the first few months after birth. In 20% - 30% of the cases, children progress to acute myeloid leukemia (AML) after remission, in which the pre-leukemic clone acquires additional mutations, such as in genes of the cohesin complex. It is hypothesized that this represents a multi-step process of leukemogenesis with three distinct genetic events: trisomy 21, GATA1 mutation and additional tertiary mutations. Here, we wanted to model the initiation and evolution of Down syndrome associated pre-leukemia and AML by employing CRISPR/Cas9. For this, we developed a CRISPR system that allows the precise manipulation of human hematopoietic stem and progenitor cells using electroporation of Cas9 protein and chemically synthesized gRNAs. We utilized human cord blood and fetal liver as a source of hematopoietic stem and progenitor cells (HSPCs). We were able to force the re-assignment of GATA1 isoforms to either the short or long isoform using CRISPR/Cas9 in purified hematopoietic stem cells (HSCs), multi-potent progenitors (MPPs), common myeloid progenitor (CMPs) and megakaryocyte/erythrocyte progenitors (MEPs). For each of these populations, we assayed their differentiation potential in single cell in vitro assays. In short, after electroporation and CRISPR/Cas9 mediated re-assignment to either the GATA1 short or long isoform, single cells were deposited onto MS5 stromal cells and were grown for 16-17 days in erythro-myeloid differentiation media. Individual colonies were analyzed by flow cytometry for their differentiation potential and genotyped to confirm CRISPR/Cas9 mediated GATA1 short or long isoform re-assignment. Overall, we were able to observe cell type specific and isoform specific effects on differentiation. For example, re-assignment to the GATA1 short isoform restricted erythroid differentiation and promoted megakaryocytic output in HSCs and MPPs. This effect was both seen when cord blood or fetal liver was used as the source of HSPCs. To confirm the role of the short isoform of GATA1, we transplanted HSCs with GATA1 short in a clonal fashion into immunocompromised mice and after 20 weeks observed grafts with high megakaryocytic output compared to control HSCs. Similarly, GlyA+ erythroid output was significantly decreased compared to transplanted control HSCs. In summary, this CRISPR/Cas9 system allows us to investigate the differentiation potential of single cells that are restricted to the endogenous expression of either the short or long isoform of GATA1. Future work will include the utilization of trisomy 21 HSCPs and the introduction of tertiary mutations, such as loss of function of STAG2, to potentially progress the model to an acute leukemia phase. Figure. Figure. Disclosures No relevant conflicts of interest to declare.