Idiopathic acquired aplastic anemia (AA) is considered an immune-mediated syndrome of bone marrow failure since approximately 70% of patients respond to immunosuppressive therapy (IST) consisting... Show moreIdiopathic acquired aplastic anemia (AA) is considered an immune-mediated syndrome of bone marrow failure since approximately 70% of patients respond to immunosuppressive therapy (IST) consisting of a course of anti-thymocyte globulin (ATG) followed by long-term use of ciclosporin. However, the immune response that underlies the pathogenesis of AA remains poorly understood. In this study, we applied high-dimensional mass cytometry on bone marrow aspirates of AA patients pre-ATG, AA patients post-ATG and healthy donors to decipher which immune cells may be implicated in the pathogenesis of AA. We show that the bone marrow of AA patients features an immune cell composition distinct from healthy donors, with significant differences in the myeloid, B-cell, CD4+ and CD8+ T-cells lineages. Specifically, we discovered that AA pre-ATG is characterized by a disease-specific immune cell network with high frequencies of CD16+ myeloid cells, CCR6++ B-cells, Th17-like CCR6+ memory CD4+ T-cells, CD45RA+CCR7+CD38+ CD8+ T-cells and KLRG1+ terminally differentiated effector memory (EMRA) CD8+ T-cells, compatible with a state of chronic inflammation. Successful treatment with IST strongly reduced the levels of CD16+ myeloid cells and showed a trend toward normalization of the frequencies of CCR6++ B-cells, CCR6+ memory CD4+ T-cells and KLRG1+EMRA CD8+ T-cells. Altogether, our study provides a unique overview of the immune landscape in bone marrow in AA at a single-cell level and proposes CCR6 as a potential new therapeutic target in AA. Show less
Human natural killer (NK) cells in lymphoid tissues can be categorized into three subsets: CD56(bright)CD16(+), CD56(dim)CD16(+) and CD69(+)CXCR6(+) lymphoid tissue-resident (lt)NK cells. How the... Show moreHuman natural killer (NK) cells in lymphoid tissues can be categorized into three subsets: CD56(bright)CD16(+), CD56(dim)CD16(+) and CD69(+)CXCR6(+) lymphoid tissue-resident (lt)NK cells. How the three subsets are functionally and developmentally related is currently unknown. Therefore, we performed single-cell RNA sequencing combined with oligonucleotide-conjugated antibodies against CD56, CXCR6, CD117 and CD34 on fresh bone marrow NK cells. A minor CD56(dim)GzmK(+) subset was identified that shared features with CD56(bright) and CD56(dim)GzmK(-) NK cells based on transcriptome, phenotype (NKG2A(high)CD16(low)KLRG1(high)TIGIT(high)) and functional analysis in bone marrow and blood, supportive for an intermediate subset. Pseudotime analysis positioned CD56(bright), CD56(dim)GzmK(+) and CD56(dim)GzmK(-) cells in one differentiation trajectory, while ltNK cells were developmentally separated. Integrative analysis with bone marrow cells from the Human Cell Atlas did not demonstrate a developmental connection between CD34(+) progenitor and NK cells, suggesting absence of early NK cell stages in bone marrow. In conclusion, single-cell transcriptomics provide new insights on development and differentiation of human NK cells. Show less
Human natural killer (NK) cells in lymphoid tissues can be categorized into three subsets: CD56(bright)CD16(+), CD56(dim)CD16(+) and CD69(+)CXCR6(+) lymphoid tissue-resident (lt)NK cells. How the... Show moreHuman natural killer (NK) cells in lymphoid tissues can be categorized into three subsets: CD56(bright)CD16(+), CD56(dim)CD16(+) and CD69(+)CXCR6(+) lymphoid tissue-resident (lt)NK cells. How the three subsets are functionally and developmentally related is currently unknown. Therefore, we performed single-cell RNA sequencing combined with oligonucleotide-conjugated antibodies against CD56, CXCR6, CD117 and CD34 on fresh bone marrow NK cells. A minor CD56(dim)GzmK(+) subset was identified that shared features with CD56(bright) and CD56(dim)GzmK(-) NK cells based on transcriptome, phenotype (NKG2A(high)CD16(low)KLRG1(high)TIGIT(high)) and functional analysis in bone marrow and blood, supportive for an intermediate subset. Pseudotime analysis positioned CD56(bright), CD56(dim)GzmK(+) and CD56(dim)GzmK(-) cells in one differentiation trajectory, while ltNK cells were developmentally separated. Integrative analysis with bone marrow cells from the Human Cell Atlas did not demonstrate a developmental connection between CD34(+) progenitor and NK cells, suggesting absence of early NK cell stages in bone marrow. In conclusion, single-cell transcriptomics provide new insights on development and differentiation of human NK cells. Show less
Human natural killer (NK) cells in lymphoid tissues can be categorized into three subsets: CD56(bright)CD16(+), CD56(dim)CD16(+) and CD69(+)CXCR6(+) lymphoid tissue-resident (lt)NK cells. How the... Show moreHuman natural killer (NK) cells in lymphoid tissues can be categorized into three subsets: CD56(bright)CD16(+), CD56(dim)CD16(+) and CD69(+)CXCR6(+) lymphoid tissue-resident (lt)NK cells. How the three subsets are functionally and developmentally related is currently unknown. Therefore, we performed single-cell RNA sequencing combined with oligonucleotide-conjugated antibodies against CD56, CXCR6, CD117 and CD34 on fresh bone marrow NK cells. A minor CD56(dim)GzmK(+) subset was identified that shared features with CD56(bright) and CD56(dim)GzmK(-) NK cells based on transcriptome, phenotype (NKG2A(high)CD16(low)KLRG1(high)TIGIT(high)) and functional analysis in bone marrow and blood, supportive for an intermediate subset. Pseudotime analysis positioned CD56(bright), CD56(dim)GzmK(+) and CD56(dim)GzmK(-) cells in one differentiation trajectory, while ltNK cells were developmentally separated. Integrative analysis with bone marrow cells from the Human Cell Atlas did not demonstrate a developmental connection between CD34(+) progenitor and NK cells, suggesting absence of early NK cell stages in bone marrow. In conclusion, single-cell transcriptomics provide new insights on development and differentiation of human NK cells. Show less
B-cell precursors (BCP) arise from hematopoietic stem cells in bone marrow (BM). Identification and characterization of the different BCP subsets has contributed to the understanding of normal B... Show moreB-cell precursors (BCP) arise from hematopoietic stem cells in bone marrow (BM). Identification and characterization of the different BCP subsets has contributed to the understanding of normal B-cell development. BCP first rearrange their immunoglobulin (Ig) heavy chain (IGH) genes to form the pre-B-cell receptor (pre-BCR) complex together with surrogate light chains. Appropriate signaling via this pre-BCR complex is followed by rearrangement of the Ig light chain genes, resulting in the formation, and selection of functional BCR molecules. Consecutive production, expression, and functional selection of the pre-BCR and BCR complexes guide the BCP differentiation process that coincides with corresponding immunophenotypic changes. We studied BCP differentiation in human BM samples from healthy controls and patients with a known genetic defect in V(D)J recombination or pre-BCR signaling to unravel normal immunophenotypic changes and to determine the effect of differentiation blocks caused by the specific genetic defects. Accordingly, we designed a 10-color antibody panel to study human BCP development in BM by flow cytometry, which allows identification of classical preB-I, preB-II, and mature B-cells as defined via BCR-related markers with further characterization by additional markers. We observed heterogeneous phenotypes associated with more than one B-cell maturation pathway, particularly for the preB-I and preB-II stages in which V(D)J recombination takes place, with asynchronous marker expression patterns. Next Generation Sequencing of complete IGH gene rearrangements in sorted BCP subsets unraveled their rearrangement status, indicating that BCP differentiation does not follow a single linear pathway. In conclusion, B-cell development in human BM is not a linear process, but a rather complex network of parallel pathways dictated by V(D)J-recombination-driven checkpoints and pre-BCR/BCR mediated-signaling occurring during B-cell production and selection. It can also be described as asynchronous, because precursor B-cells do not differentiate as full population between the different stages, but rather transit as a continuum, which seems influenced (in part) by V-D-J recombination-driven checkpoints. Show less
Faraci, M.; Bertaina, A.; Dalissier, A.; Ifversen, M.; Schulz, A.; Gennery, A.; ... ; EBMT Pediat Dis Working Party 2019
BM has been put forward as a major reservoir for memory CD8(+) T cells. In order to fulfill that function, BM should "store" memory CD8(+) T cells, which in biological terms would require these ... Show moreBM has been put forward as a major reservoir for memory CD8(+) T cells. In order to fulfill that function, BM should "store" memory CD8(+) T cells, which in biological terms would require these "stored" memory cells to be in disequilibrium with the circulatory pool. This issue is a matter of ongoing debate. Here, we unequivocally demonstrate that murine and human BM harbors a population of tissue-resident memory CD8(+) T (T-RM) cells. These cells develop against various pathogens, independently of BM infection or local antigen recognition. BM CD8(+) T-RM cells share a transcriptional program with resident lymphoid cells in other tissues; they are polyfunctional cytokine producers and dependent on IL-15, Blimp-1, and Hobit. CD8(+) T-RM cells reside in the BM parenchyma, but are in close contact with the circulation. Moreover, this pool of resident T cells is not size-restricted and expands upon peripheral antigenic re-challenge. This works extends the role of the BM in the maintenance of CD8(+) T cell memory to include the preservation of an expandable reservoir of functional, non-recirculating memory CD8(+) T cells, which develop in response to a large variety of peripheral antigens. Show less
