Secreted factors from mouse embryonic fibroblasts maintain repopulating function of single cultured hematopoietic stem cells.

Hematopoietic stem cell self-renewal, proliferation, and differentiation are independently regulated by intrinsic as well as extrinsic mechanisms. We previously demonstrated that murine proliferation of hematopoietic stem cells is supported in serum-free medium supplemented with two growth factors, stem cell factor and interleukin 11. The survival of hematopoietic stem cells is additionally improved by supplementing this medium with two more growth factors, neural growth factor and collagen 1 (four growth factors) or serum-free medium conditioned by the hematopoietic stem cell-supportive stromal UG26-1B6 cells1. Here, we describe a robust and versatile alternative source of conditioned medium from mouse embryonic fibroblasts. We found that this conditioned medium supports survival and phenotypical identity of hematopoietic stem cells, as well as cell cycle entry in single cell cultures of CD34- CD48- CD150+ Lineage- SCA1+ KIT+ cells supplemented with two growth factors. Strikingly, in comparison with cultures in serum-free medium with four growth factors, conditioned medium from mouse embryonic fibroblasts increases the numbers of proliferating clones and the number of Lineage- SCA1+ KIT+ cells, both with two and four growth factors. In addition, conditioned medium from mouse embryonic fibroblasts supports self-renewal in culture of cells with short- and long-term hematopoiesis-repopulating ability in vivo. These findings identify conditioned medium from mouse embryonic fibroblasts as a robust alternative serumfree source of factors to maintain self-renewal of in vivo-repopulating hematopoetic stem cells in culture.

An Evaluation of T-Cell Functionality After Flow Cytometry Sorting Revealed p38 MAPK Activation.

Cell alterations during isolation and preparation for flow cytometry cell sorting by antibodies, temperature, homogenization, buffer composition and mitogens are well known. In contrast, little is known about cell alteration caused by the instrument or the sorting process itself. We systematically evaluated cellular responses to different sorter-induced physical forces. In summary, flow cytometry cell-sorting induced forces can affect cellular signaling cascades, especially the MAPK p38. Functional assays, related to the p38 MAPK pathway, of human primary T cells after flow cytometry sorting did lead to minor physiological modulation but no functional impairments. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Niche WNT5A regulates the actin cytoskeleton during regeneration of hematopoietic stem cells.

Here, we show that the Wnt5a-haploinsufficient niche regenerates dysfunctional HSCs, which do not successfully engraft in secondary recipients. RNA sequencing of the regenerated donor Lin- SCA-1+ KIT+ (LSK) cells shows dysregulated expression of ZEB1-associated genes involved in the small GTPase-dependent actin polymerization pathway. Misexpression of DOCK2, WAVE2, and activation of CDC42 results in apolar F-actin localization, leading to defects in adhesion, migration and homing of HSCs regenerated in a Wnt5a-haploinsufficient microenvironment. Moreover, these cells show increased differentiation in vitro, with rapid loss of HSC-enriched LSK cells. Our study further shows that the Wnt5a-haploinsufficient environment similarly affects BCR-ABLp185 leukemia-initiating cells, which fail to generate leukemia in 42% of the studied recipients, or to transfer leukemia to secondary hosts. Thus, we show that WNT5A in the bone marrow niche is required to regenerate HSCs and leukemic cells with functional ability to rearrange the actin cytoskeleton and engraft successfully.

CD25+ FoxP3+ Memory CD4 T Cells Are Frequent Targets of HIV Infection In Vivo.

UNLABELLED: Interleukin 2 (IL-2) signaling through the IL-2 receptor alpha chain (CD25) facilitates HIV replication in vitro and facilitates homeostatic proliferation of CD25(+) FoxP3(+) CD4(+) T cells. CD25(+) FoxP3(+) CD4(+) T cells may therefore constitute a suitable subset for HIV infection and plasma virion production. CD25(+) FoxP3(+) CD4(+) T cell frequencies, absolute numbers, and the expression of CCR5 and cell cycle marker Ki67 were studied in peripheral blood from HIV(+) and HIV(-) study volunteers. Different memory CD4(+) T cell subsets were then sorted for quantification of cell-associated HIV DNA and phylogenetic analyses of the highly variable EnvV1V3 region in comparison to plasma-derived virus sequences. In HIV(+) subjects, 51% (median) of CD25(+) FoxP3(+) CD4(+) T cells expressed the HIV coreceptor CCR5. Very high frequencies of Ki67(+) cells were detected in CD25(+) FoxP3(+) memory CD4(+) T cells (median, 27.6%) in comparison to CD25(-) FoxP3(-) memory CD4(+) T cells (median, 4.1%; P < 0.0001). HIV DNA content was 15-fold higher in CD25(+) FoxP3(+) memory CD4(+) T cells than in CD25(-) FoxP3(-) T cells (P = 0.003). EnvV1V3 sequences derived from CD25(+) FoxP3(+) memory CD4(+) T cells did not preferentially cluster with plasma-derived sequences. Quasi-identical cell-plasma sequence pairs were rare, and their proportion decreased with the estimated HIV infection duration. These data suggest that specific cellular characteristics of CD25(+) FoxP3(+) memory CD4(+) T cells might facilitate efficient HIV infection in vivo and passage of HIV DNA to cell progeny in the absence of active viral replication. The contribution of this cell population to plasma virion production remains unclear. IMPORTANCE: Despite recent advances in the understanding of AIDS virus pathogenesis, which cell subsets support HIV infection and replication in vivo is incompletely understood. In vitro, the IL-2 signaling pathway and IL-2-dependent cell cycle induction are essential for HIV infection of stimulated T cells. CD25(+) FoxP3(+) memory CD4 T cells, often referred to as regulatory CD4 T cells, depend on IL-2 signaling for homeostatic proliferation in vivo Our results show that CD25(+) FoxP3(+) memory CD4(+) T cells often express the HIV coreceptor CCR5, are significantly more proliferative, and contain more HIV DNA than CD25(-) FoxP3(-) memory CD4 T cell subsets. The specific cellular characteristics of CD25(+) FoxP3(+) memory CD4(+) T cells probably facilitate efficient HIV infection in vivo and passage of HIV DNA to cell progeny in the absence of active viral replication. However, the contribution of this cell subset to plasma viremia remains unclear.

Stroma-Derived Connective Tissue Growth Factor Maintains Cell Cycle Progression and Repopulation Activity of Hematopoietic Stem Cells In Vitro.

Hematopoietic stem cells (HSCs) are preserved in co-cultures with UG26-1B6 stromal cells or their conditioned medium. We performed a genome-wide study of gene expression changes of UG26-1B6 stromal cells in contact with Lineage⁻ SCA-1⁺ KIT⁺ (LSK) cells. This analysis identified connective tissue growth factor (CTGF) to be upregulated in response to LSK cells. We found that co-culture of HSCs on CTGF knockdown stroma (shCtgf) shows impaired engraftment and long-term quality. Further experiments demonstrated that CD34⁻ CD48⁻ CD150⁺ LSK (CD34⁻ SLAM) cell numbers from shCtgf co-cultures increase in G0 and senescence and show delayed time to first cell division. To understand this observation, a CTGF signaling network model was assembled, which was experimentally validated. In co-culture experiments of CD34⁻ SLAM cells with shCtgf stromal cells, we found that SMAD2/3-dependent signaling was activated, with increasing p27(Kip1) expression and downregulating cyclin D1. Our data support the view that LSK cells modulate gene expression in the niche to maintain repopulating HSC activity.

Serial transfer of single-cell-derived immunocompetence reveals stemness of CD8(+) central memory T cells.

Maintenance of immunological memory has been proposed to rely on stem-cell-like lymphocytes. However, data supporting this hypothesis are focused on the developmental potential of lymphocyte populations and are thus insufficient to establish the functional hallmarks of stemness. Here, we investigated self-renewal capacity and multipotency of individual memory lymphocytes by in vivo fate mapping of CD8(+) T cells and their descendants across three generations of serial single-cell adoptive transfer and infection-driven re-expansion. We found that immune responses derived from single naive T (Tn) cells, single primary, and single secondary central memory T (Tcm) cells reached similar size and phenotypic diversity, were subjected to comparable stochastic variation, and could ultimately reconstitute immunocompetence against an otherwise lethal infection with the bacterial pathogen Listeria monocytogenes. These observations establish that adult tissue stem cells reside within the CD62L(+) Tcm cell compartment and highlight the promising therapeutic potential of this immune cell subset.

MHC multimer-guided and cell culture-independent isolation of functional T cell receptors from single cells facilitates TCR identification for immunotherapy.

Adoptive therapy using T cells redirected to target tumor- or infection-associated antigens is a promising strategy that has curative potential and broad applicability. In order to accelerate the screening process for suitable antigen-specific T cell receptors (TCRs), we developed a new approach circumventing conventional in vitro expansion-based strategies. Direct isolation of paired full-length TCR sequences from non-expanded antigen-specific T cells was achieved by the establishment of a highly sensitive PCR-based T cell receptor single cell analysis method (TCR-SCAN). Using MHC multimer-labeled and single cell-sorted HCMV-specific T cells we demonstrate a high efficacy (approximately 25%) and target specificity of TCR-SCAN receptor identification. In combination with MHC-multimer based pre-enrichment steps, we were able to isolate TCRs specific for the oncogenes Her2/neu and WT1 even from very small populations (original precursor frequencies of down to 0.00005% of CD3(+) T cells) without any cell culture step involved. Genetic re-expression of isolated receptors demonstrates their functionality and target specificity. We believe that this new strategy of TCR identification may provide broad access to specific TCRs for therapeutically relevant T cell epitopes.

CXC chemokine receptor 4 is essential for maintenance of renal cell carcinoma-initiating cells and predicts metastasis.

In many solid tumors, cancer stem cells (CSC) represent a population with tumor-initiating, self-renewal, and differentiation potential, which can be identified by surface protein markers. No generally applicable markers are yet known for renal cell carcinoma (RCC). Two RCC cell lines (RCC-26, RCC-53) were found to differ widely in their capacity to form spheres in vitro and to establish tumors in mice, potentially reflecting differences in CSC content. A subpopulation expressing the CXC chemokine receptor 4 (CXCR4) was present only in the more tumorigenic cell line RCC-53. When grown as spheres, most of the RCC-53 cells were CXCR4-positive, expressed stem cell-associated transcription factor genes at elevated levels, and were more resistant toward the tyrosine kinase inhibitors sunitinib, sorafenib, and pazopanib. Sorted CXCR4-positive cells exhibited greater capacity for sphere formation and tumor growth-inducing potential in vivo than CXCR4-negative cells. Significantly, higher CXCR4 mRNA levels in primary RCC tumors from patients with localized but not disseminated disease predicted shorter survival. Downregulation of CXCR4 expression by small interfering RNA (siRNA) or pharmacological inhibition by AMD3100 compromised tumor sphere formation, viability of CXCR4-positive cells, and increased their responsiveness toward tyrosine kinase inhibitors. In conclusion, CXCR4 identifies a subpopulation of tumor-initiating cells in RCC cell lines and plays a role in their maintenance. The relative insensitivity of such cells to tyrosine kinase inhibitors might contribute to the development of therapy resistance in RCC patients. Future therapies therefore could combine blockade of the CXCR4 signaling pathway with standard therapies for more effective treatments of metastatic RCC.

Identification of CCR9- murine plasmacytoid DC precursors with plasticity to differentiate into conventional DCs.

Whereas the final differentiation of conventional dendritic cells (CDCs) from committed precursors occurs locally in secondary lymphoid or peripheral tissues, plasmacytoid dendritic cells (PDCs) are thought to fully develop in the bone marrow from common DC progenitors before migrating to the periphery. In our study, we define, for the first time, a subpopulation of CCR9(-) major histocompatibility complex class II(low) PDCs in murine bone marrow, which express E2-2 and are immediate precursors of CCR9(+) fully differentiated PDCs. However, CCR9(-) PDCs have the plasticity to acquire the phenotype and function of CD11b(+) CD8α(-) major histocompatibility complex class II(high) CDC-like cells under the influence of soluble factors produced by intestinal epithelial cells or recombinant GM-CSF. This deviation from the PDC lineage commitment is regulated on the level of transcription factors reflected by down-regulation of E2-2 and up-regulation of ID2, PU.1, and BATF3. Thus, CCR9(-) PDCs are immediate PDC precursors that can be reprogrammed to differentiate into CDC-like cells with higher antigen-presenting and cytokine-producing capacity under the influence of the local tissue microenvironment.