Therapeutic gene editing of T cells to correct CTLA-4 insufficiency.

Heterozygous mutations in CTLA-4 result in an inborn error of immunity with an autoimmune and frequently severe clinical phenotype. Autologous T cell gene therapy may offer a cure without the immunological complications of allogeneic hematopoietic stem cell transplantation. Here, we designed a homology-directed repair (HDR) gene editing strategy that inserts the CTLA-4 cDNA into the first intron of the CTLA-4 genomic locus in primary human T cells. This resulted in regulated expression of CTLA-4 in CD4+ T cells, and functional studies demonstrated CD80 and CD86 transendocytosis. Gene editing of T cells isolated from three patients with CTLA-4 insufficiency also restored CTLA-4 protein expression and rescued transendocytosis of CD80 and CD86 in vitro. Last, gene-corrected T cells from CTLA-4-/- mice engrafted and prevented lymphoproliferation in an in vivo murine model of CTLA-4 insufficiency. These results demonstrate the feasibility of a therapeutic approach using T cell gene therapy for CTLA-4 insufficiency.

Dissecting pathways to thrombocytopenia in a mouse model of visceral leishmaniasis.

Visceral leishmaniasis is an important yet neglected parasitic disease caused by infection with Leishmania donovani or L infantum. Disease manifestations include fever, weight loss, hepatosplenomegaly, immune dysregulation, and extensive hematological complications. Thrombocytopenia is a dominant hematological feature seen in both humans and experimental models, but the mechanisms behind this infection-driven thrombocytopenia remain poorly understood. Using a murine model of experimental visceral leishmaniasis (EVL), we demonstrated a progressive decrease in platelets from day 14 after infection, culminating in severe thrombocytopenia by day 28. Plasma thrombopoietin (TPO) levels were reduced in infected mice, at least in part because of the alterations in the liver microenvironment associated with granulomatous inflammation. Bone marrow (BM) megakaryocyte cytoplasmic maturation was significantly reduced. In addition to a production deficit, we identified significant increases in platelet clearance. L donovani-infected splenectomized mice were protected from thrombocytopenia compared with sham operated infected mice and had a greater response to exogenous TPO. Furthermore, infection led to higher levels of platelet opsonization and desialylation, both associated with platelet clearance in spleen and liver, respectively. Critically, these changes could be reversed rapidly by drug treatment to reduce parasite load or by administration of TPO agonists. In summary, our findings demonstrate that the mechanisms underpinning thrombocytopenia in EVL are multifactorial and reversible, with no obvious residual damage to the BM microenvironment.

CD4+ T Cells Alter the Stromal Microenvironment and Repress Medullary Erythropoiesis in Murine Visceral Leishmaniasis.

Human visceral leishmaniasis, a parasitic disease of major public health importance in developing countries, is characterized by variable degrees of severity of anemia, but the mechanisms underlying this change in peripheral blood have not been thoroughly explored. Here, we used an experimental model of visceral leishmaniasis in C57BL/6 mice to explore the basis of anemia following infection with Leishmania donovani. 28 days post-infection, mice showed bone marrow dyserythropoiesis by myelogram, with a reduction of TER119+ CD71-/+ erythroblasts. Reduction of medullary erythropoiesis coincided with loss of CD169high bone marrow stromal macrophages and a reduction of CXCL12-expressing stromal cells. Although the spleen is a site of extramedullary erythropoiesis and erythrophagocytosis, splenectomy did not impact the extent of anemia or affect the repression of medullary hematopoiesis that was observed in infected mice. In contrast, these changes in bone marrow erythropoiesis were not evident in B6.Rag2-/- mice, but could be fully reconstituted by adoptive transfer of IFNγ-producing but not IFNγ-deficient CD4+ T cells, mimicking the expansion of IFNγ-producing CD4+ T cells that occurs during infection in wild type mice. Collectively, these data indicate that anemia during experimental murine visceral leishmaniasis can be driven by defects associated with the bone marrow erythropoietic niche, and that this represents a further example of CD4+ T cell-mediated immunopathology affecting hematopoietic competence.

TNF signalling drives expansion of bone marrow CD4+ T cells responsible for HSC exhaustion in experimental visceral leishmaniasis.

Visceral leishmaniasis is associated with significant changes in hematological function but the mechanisms underlying these changes are largely unknown. In contrast to naïve mice, where most long-term hematopoietic stem cells (LT-HSCs; LSK CD150+ CD34- CD48- cells) in bone marrow (BM) are quiescent, we found that during Leishmania donovani infection most LT-HSCs had entered cell cycle. Loss of quiescence correlated with a reduced self-renewal capacity and functional exhaustion, as measured by serial transfer. Quiescent LT-HSCs were maintained in infected RAG2 KO mice, but lost following adoptive transfer of IFNγ-sufficient but not IFNγ-deficient CD4+ T cells. Using mixed BM chimeras, we established that IFNγ and TNF signalling pathways converge at the level of CD4+ T cells. Critically, intrinsic TNF signalling is required for the expansion and/or differentiation of pathogenic IFNγ+CD4+ T cells that promote the irreversible loss of BM function. These findings provide new insights into the pathogenic potential of CD4+ T cells that target hematopoietic function in leishmaniasis and perhaps other infectious diseases where TNF expression and BM dysfunction also occur simultaneously.

Multiparameter Analysis of Human Bone Marrow Stromal Cells Identifies Distinct Immunomodulatory and Differentiation-Competent Subtypes.

Bone marrow stromal cells (BMSCs, also called bone-marrow-derived mesenchymal stromal cells) provide hematopoietic support and immunoregulation and contain a stem cell fraction capable of skeletogenic differentiation. We used immortalized human BMSC clonal lines for multi-level analysis of functional markers for BMSC subsets. All clones expressed typical BMSC cell-surface antigens; however, clones with trilineage differentiation capacity exhibited enhanced vascular interaction gene sets, whereas non-differentiating clones were uniquely CD317 positive with significantly enriched immunomodulatory transcriptional networks and high IL-7 production. IL-7 lineage tracing and CD317 immunolocalization confirmed the existence of a rare non-differentiating BMSC subtype, distinct from Cxcl12-DsRed(+) perivascular stromal cells in vivo. Colony-forming CD317(+) IL-7(hi) cells, identified at ∼ 1%-3% frequency in heterogeneous human BMSC fractions, were found to have the same biomolecular profile as non-differentiating BMSC clones using Raman spectroscopy. Distinct functional identities can be assigned to BMSC subpopulations, which are likely to have specific roles in immune control, lymphopoiesis, and bone homeostasis.