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To identify the differences between aged and young human hematopoiesis, we performed a direct comparison of aged and young human hematopoietic stem and progenitor cells (HSPCs). Alterations in transcriptome profiles upon aging between humans and mice were then compared. Human specimens consist of CD34+ cells from bone marrow, and mouse specimens of hematopoietic stem cells (HSCs; Lin- Kit+ Sca1+ CD150+). Single-cell transcriptomic studies, functional clustering, and developmental trajectory analyses were performed. A significant increase in multipotent progenitor 2A (MPP2A) cluster is found in the early HSC trajectory in old human subjects. This cluster is enriched in senescence signatures (increased telomere attrition, DNA damage, activation of P53 pathway). In mouse models, the accumulation of an analogous subset was confirmed in the aged LT-HSC population. Elimination of this subset has been shown to rejuvenate hematopoiesis in mice. A significant activation of the P53-P21WAF1/CIP1 pathway was found in the MPP2A population in humans. In contrast, the senescent HSCs in mice are characterized by activation of the p16Ink4a pathway. Aging in the human HSC compartment is mainly caused by the clonal evolution and accumulation of a senescent cell cluster. A population with a similar senescence signature in the aged LT-HSCs was confirmed in the murine aging model. Clearance of this senescent population with senotherapy in humans is feasible and potentially beneficial.

Adhesion of hematopoietic stem and progenitor cells (HSPCs) to the bone marrow niche plays critical roles in the maintenance of the most primitive HSPCs. The interactions of HSPC-niche interactions are clinically relevant in acute myeloid leukemia (AML), because (i) leukemia-initiating cells adhered to the marrow niche are protected from the cytotoxic effect by chemotherapy and (ii) mobilization of HSPCs from healthy donors' bone marrow is crucial for the effective stem cell transplantation. However, although many clinical agents have been developed for the HSPC mobilization, the effects caused by the extrinsic molecular cues were traditionally evaluated based on phenomenological observations. This review highlights the recent interdisciplinary challenges of hematologists, biophysicists and cell biologists towards the design of defined in vitro niche models and the development of physical biomarkers for quantitative indexing of differential effects of clinical agents on human HSPCs., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Chronic kidney failure (KF) provokes the development of immune senescent CD8 + cytotoxic T cells, affecting the occurrence of graft rejection, viral infections, and malignancies after kidney transplantation. In this study, we analyzed the impact of KF, subsequent dialysis treatment, and kidney transplantation on the differentiation of CD8 + CD31 + CD45RA + CCR7 + recent thymic emigrant (CCR7 + RTE) Tregs/Tresps into CD8 + CD31 - CD45RA - memory (CD31 - memory) Tregs/Tresps and its effect on the release of cytokines, Fas receptor, Fas ligand as well as cytotoxic mediators by naïve, central memory (CM), effector memory (EM), and terminally differentiated effector memory (TEMRA) Tresps. We found that normal age-dependent differentiation of CD8 + Tregs/Tresps generally differs in the way that TEMRA cells only arise in Tresps. Compared to healthy controls, KF patients revealed an age-independently decreased frequency of CCR7 + RTE Tregs/Tresps, but increased frequencies of CCR7 + MN Tregs/Tresps and CD31 - memory Tregs/Tresps, suggesting an increased differentiation via CD31 + CD45RA - memory (CD31 + memory) Tregs/Tresps into CD31 - memory Tregs/Tresps. Intensified differentiation via CD31 + memory Tresps increased the emergence of apoptosis-resistant CM Tresps with strong Fas ligand-mediated cytotoxicity. CCR7 + RTE Tresp proliferation generated TEMRA Tresps, secreting high levels of cytotoxic mediators. In dialysis and transplant patients, CD31 + TEMRA Tregs/Tresps accumulated, proposing an impaired CCR7 + RTE Treg/Tresp differentiation via CD31 + memory Tregs/Tresps into CD31 - memory Tregs/Tresps. Increased percentages of CD31 - TEMRA Tresps, but not of CD31 - TEMRA Tregs, were observed in all patient groups, indicating impaired proliferation of CCR7 + RTE Tresps, but not of CCR7 + RTE Tregs, into CD31 - memory Tregs/Tresps. In transplant patients, CCR7 + RTE Tregs accumulated, while frequencies of CCR7 + RTE Tresps were decreased, suggesting that the immunosuppressive therapy only prevented excessive CCR7 + RTE Treg differentiation but not that of CCR7 + RTE Tresps. Presumably, this caused the accumulation of TEMRA Tresps with decreased release of cytotoxic mediators, such as perforin. In conclusion, we propose that chronic KF affects both the differentiation of CD8 + Tregs and CD8 + Tresps. However, the immunosuppressive therapy after transplantation may successfully prevent excessive Treg differentiation, but not as suffciently that of Tresps. Therefore, the risk for graft rejection may be reduced, while the susceptibility for infections and malignancies may be increased in these patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Leonhard, Schaier, Kälble, Eckstein, Zeier and Steinborn.)

Elimination of suppressive T cells may enable and enhance cancer immunotherapy. Here, we demonstrate that the cell membrane protein SLAMF7 was highly expressed on immunosuppressive CD8 + CD28 - CD57 + Tregs in multiple myeloma (MM). SLAMF7 expression associated with T cell exhaustion surface markers and exhaustion-related transcription factor signatures. T cells from patients with a high frequency of SLAMF7 + CD8 + T cells exhibited decreased immunoreactivity towards the MART-1 aa26-35*A27L antigen. A monoclonal anti-SLAMF7 antibody (elotuzumab) specifically depleted SLAMF7 + CD8 + T cells in vitro and in vivo via macrophage-mediated antibody-dependent cellular phagocytosis (ADCP). Anti-SLAMF7 treatment of MM patients depleted suppressive T cells in peripheral blood. These data highlight SLAMF7 as a marker for suppressive CD8 + Treg and suggest that anti-SLAMF7 antibodies can be used to boost anti-tumoral immune responses in cancer patients., (© 2021. The Author(s).)

To acquire a better understanding of clonal evolution of acute myeloid leukemia (AML) and to identify the clone(s) responsible for disease recurrence, we have comparatively studied leukemia-specific mutations by whole-exome-sequencing (WES) of both the leukemia and the nonleukemia compartments derived from the bone marrow of AML patients. The T-lymphocytes, B-lymphocytes and the functionally normal hematopoietic stem cells (HSC), that is, CD34 + /CD38 - /ALDH + cells for AML with rare-ALDH + blasts (<1.9% ALDH + cells) were defined as the nonleukemia compartments. WES identified 62 point-mutations in the leukemia compartment derived from 12 AML-patients at the time of diagnosis and 73 mutations in 3 matched relapse cases. Most patients (8/12) showed 4 to 6 point-mutations per sample at diagnosis. Other than the mutations in the recurrently mutated genes such as DNMT3A, NRAS and KIT, we were able to identify novel point-mutations that have not yet been described in AML. Some leukemia-specific mutations and cytogenetic abnormalities including DNMT3A(R882H), EZH2(I146T) and inversion(16) were also detectable in the respective T-lymphocytes, B-lymphocytes and HSC in 5/12 patients, suggesting that preleukemia HSC might represent the source of leukemogenesis for these cases. The leukemic evolution was reconstructed for five cases with detectable preleukemia clones, which were tracked in follow-up and relapse samples. Four of the five patients with detectable preleukemic mutations developed relapse. The presence of leukemia-specific mutations in these nonleukemia compartments, especially after chemotherapy or after allogeneic stem cell transplantation, is highly relevant, as these could be responsible for relapse. This discovery may facilitate the identification of novel targets for long-term cure., (© 2021 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Chimeric antigen receptor T (CAR-T) cells targeting CD19 came into clinical practice for the treatment of B cell lymphoma in 2018. However, patients being treated for B cell lymphoma often suffer from comorbidities such as chronic pain, cardiovascular diseases and arthritis. Thus, these patients frequently receive concomitant medications that include nonsteroidal anti-inflammatory drugs (NSAIDs) like cyclooxygenase (COX) inhibitors. Celecoxib, a selective COX-2 inhibitor, and aspirin, a non-selective COX-1 and COX-2 inhibitor, are being used as anti-inflammatory, analgesic and anti-pyretic drugs. In addition, several studies have also focused on the anti-neoplastic properties of COX-inhibitors. As the influence of COX-inhibitors on CD19.CAR-T cells is still unknown, we investigated the effect of celecoxib and aspirin on the quantity and quality of CD19.CAR-T cells at different concentrations with special regard to cytotoxicity, activation, cytokine release, proliferation and exhaustion. A significant effect on CAR-T cells could be observed for 0.1 mmol/L of celecoxib and for 4 mmol/L of aspirin. At these concentrations, we found that both COX-inhibitors could induce intrinsic apoptosis of CD19.CAR-T cells showing a significant reduction in the ratio of JC-10 red to JC-10 green CAR-T cells from 6.46 ± 7.03 (mean ± SD) to 1.76 ± 0.67 by celecoxib and to 4.41 ± 0.32 by aspirin, respectively. Additionally, the ratios of JC-10 red to JC-10 green Daudi cells were also decreased from 3.41 ± 0.30 to 0.77 ± 0.06 by celecoxib and to 1.26 ± 0.04 by aspirin, respectively. Although the cytokine release by CD19.CAR-T cells upon activation was not hampered by both COX-inhibitors, activation and proliferation of CAR-T cells were significantly inhibited via diminishing the NF-ĸB signaling pathway by a significant down-regulation of expression of CD27 on CD4 + and CD8 + CAR-T cells, followed by a clear decrease of phosphorylated NF-ĸB p65 in both CD4 + and CD8 + CAR-T cells by a factor of 1.8. Of note, COX-inhibitors hampered expansion and induced exhaustion of CAR-T cells in an antigen stress assay. Collectively, our findings indicate that the use of COX-inhibitors is a double-edged sword that not only induces apoptosis in tumor cells but also impairs the quantity and quality of CAR-T cells. Therefore, COX-inhibitors should be used with caution in patients with B cell lymphoma under CAR-T cell therapy., Competing Interests: MS received funding for collaborative research from Apogenix, Hexal and Novartis, travel grants from Hexal and Kite, he received financial support for educational activities and conferences from bluebird bio, Kite and Novartis, he is a board member for MSD and (co-)PI of clinical trials of MSD, GSK, Kite and BMS, as well as co-founder and shareholder of TolerogenixX Ltd. AS received travel grants from Hexal and Jazz Pharmaceuticals, research grant from Therakos/Mallinckrodt and is co-founder of TolerogenixX Ltd. AS and LW are part- or full-time employers of TolerogenixX Ltd. LS was employed by Takeda Pharma Vertrieb GmbH & Co. KG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yang, Wang, Ni, Neuber, Wang, Gong, Sauer, Schubert, Hückelhoven-Krauss, Xia, Ge, Kleist, Eckstein, Sellner, Müller-Tidow, Dreger, Schmitt and Schmitt.)

Objectives: An imbalance between CD4+-regulatory T-cells (Tregs) and CD4+-responder T-cells (Tresps) correlates with active disease flares in systemic lupus erythematosus (SLE) patients. Both cell subsets consist of highly proliferating Tregs/Tresps expressing inducible T-cell co-stimulatory molecule (ICOS) and less proliferating ICOS--Tregs/Tresps., Methods: Six-colour-flow-cytometric analysis was used to examine the effect of ICOS+- and ICOS--Treg/Tresp cell differentiation on the composition of the total CD4+-T-helper cell pool with ICOS+- and ICOS--Tregs/Tresps. Functionality of Tregs was examined using suppression assays., Results: In 83 healthy volunteers, the ratio of ICOS+-Tregs/ICOS+-Tresps increased significantly with age, while that of ICOS--Tregs/ICOS--Tresps did not change. In 86 SLE patients (SLEDAI <7), disease activity was associated with an age-independently increased ratio of both ICOS+-Tregs/ICOS+-Tresps and ICOS--Tregs/ICOS--Tresps. In these patients, the functional activity of ICOS+-Tregs, but not of ICOS--Tregs, was preserved. In 13 markedly active disease patients (SLEDAI >7), the percentage of both ICOS+-Tregs and ICOS+-Tresps, was strongly increased within total CD4+-T-helper cells. However, the increased ratio of ICOS+-Tregs/ICOS+-Tresps was not maintained in these patients, due to terminal differentiation and accumulation of naïve cells within total ICOS+-Tregs. Despite increased differentiation of both ICOS--Tregs and ICOS--Tresps, the percentage of ICOS--Tregs increased within CD4+-T-helper cells, while that of ICOS--Tresps decreased, resulting in a significantly increased ratio of ICOS--Tregs/ICOS--Tresps independent of age., Conclusions: Our data reveal a crucial role of Treg immune senescence for the occurrence of disease flares in SLE patients, with ICOS+-Treg cells being most affected.

SHANK2 mutations have been identified in individuals with neurodevelopmental disorders, including intellectual disability and autism spectrum disorders (ASD). Using CRISPR/Cas9 genome editing, we obtained SH-SY5Y cell lines with frameshift mutations on one or both SHANK2 alleles. We investigated the effects of the different SHANK2 mutations on cell morphology, cell proliferation and differentiation potential during early neuronal differentiation. All mutant cell lines showed impaired neuronal differentiation marker expression. Cells with bi-allelic SHANK2 mutations revealed diminished apoptosis and increased proliferation, as well as decreased neurite outgrowth during early neuronal differentiation. Bi-allelic SHANK2 mutations resulted in an increase in p-AKT levels, suggesting that SHANK2 mutations impair downstream signaling of tyrosine kinase receptors. Additionally, cells with bi-allelic SHANK2 mutations had lower amyloid precursor protein (APP) expression compared to controls, suggesting a molecular link between SHANK2 and APP. Together, we can show that frameshift mutations on one or both SHANK2 alleles lead to an alteration of neuronal differentiation in SH-SY5Y cells, characterized by changes in cell growth and pre- and postsynaptic protein expression. We also provide first evidence that downstream signaling of tyrosine kinase receptors and amyloid precursor protein expression are affected.

Chimeric antigen receptor (CAR) T cell therapy has shown promising responses in patients with refractory or relapsed aggressive B-cell malignancies that are resistant to conventional chemotherapy or stem cell transplantation. A potentially combinatorial therapeutic strategy may be the inhibition of anti-apoptotic Bcl-2 family proteins, overexpressed in most cancer cells. In this study we investigated the combination of 3rd-generation CD19.CAR-T cells and the BH3 mimetics venetoclax, a Bcl-2 inhibitor, or S63845, a Mcl-1 inhibitor, under three different treatment conditions: pre-sensitization of cancer cells with BH3 mimetics followed by CAR-T cell treatment, simultaneous combination therapy, and the administration of BH3 mimetics after CAR-T cell treatment. Our results showed that administration of CAR-T cells and BH3 mimetics had a significant effect on the quantity and quality of CD19.CAR-T cells. The administration of BH3 mimetics prior to CAR-T cell therapy exerted an enhanced cytotoxic efficacy by upregulating the CD19 expression and pro-apoptotic proteins in highly sensitive tumor cells, and thereby improving both CD19.CAR-T cell cytotoxicity and persistence. In simultaneous and post-treatment approaches, however, the quantity of CAR-T cells was adversely affected. Our findings indicate pre-sensitization of highly sensitive tumor cells with BH3 mimetics could enhance the cytotoxic efficacy of CAR-T cell treatment., Competing Interests: MS received funding for collaborative research from Apogenix, Hexal and Novartis, travel grants from Hexal and Kite, received financial support for educational activities and conferences from bluebird bio, Kite and Novartis, is a board member for MSD and (co-)PI of clinical trials of MSD, GSK, Kite, and BMS, as well as a co-founder and shareholder of TolerogenixX Ltd. AS received travel grants from Hexal and Jazz Pharmaceuticals, research grant from Therakos/Mallinckrodt, and is a co-founder of TolerogenixX LtD. AS and LW are part- or full-time employers of TolerogenixX Ltd. LS was employed by Takeda Pharma Vertrieb GmbH & Co. KG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a past collaboration with one of the authors TS., (Copyright © 2020 Yang, Wang, Ni, Neuber, Wang, Gong, Sauer, Sellner, Schubert, Hückelhoven-Krauss, Hong, Zhu, Kleist, Eckstein, Müller-Tidow, Dreger, Schmitt and Schmitt.)

Background: CD4 + T cells are of great importance in the pathogenesis of systemic lupus erythematosus (SLE), as an imbalance between CD4 + regulatory T cells (Tregs) and CD4 + responder T cells (Tresps) causes flares of active disease in SLE patients. In this study, we aimed to find the role of aberrant Treg/Tresp cell differentiation for maintaining Treg/Tresp cell balance and Treg functionality., Methods: To determine differences in the differentiation of Tregs/Tresps we calculated the percentages of CD45RA + CD31 + recent thymic emigrant (RTE) Tregs/Tresps and CD45RA + CD31 - mature naive (MN) Tregs/Tresps, as well as CD45RA - CD31 + and CD45RA - CD31 - memory Tregs/Tresps (CD31 + and CD31 - memory Tregs/Tresps) within the total Treg/Tresp pool of 78 SLE remission patients compared with 94 healthy controls of different ages. The proliferation capacity of each Treg/Tresp subset was determined by staining the cells with anti-Ki67 monoclonal antibodies. Differences in the autologous or allogeneic Treg function between SLE remission patients and healthy controls were determined using suppression assays., Results: With age, we found an increased differentiation of RTE Tregs via CD31 + memory Tregs and of RTE Tresps via MN Tresps into CD31 - memory Tregs/Tresp in healthy volunteers. This opposite differentiation of RTE Tregs and Tresps was associated with an age-dependent increase in the suppressive activity of both naive and memory Tregs. SLE patients showed similar age-dependent Treg cell differentiation. However, in these patients RTE Tresps differentiated increasingly via CD31 + memory Tresps, whereby CD31 - memory Tresps arose that were much more difficult to inhibit for Tregs than those that emerged through differentiation via MN Tresps. Consequently, the increase in the suppressive activity of Tregs with age could not be maintained in SLE patients. Testing the Tregs of healthy volunteers and SLE patients with autologous and nonautologous Tresps revealed that the significantly decreased Treg function in SLE patients was not exclusively attributed to an age-dependent diminished sensitivity of the Tresps for Treg suppression. The immunosuppressive therapy reduced the accelerated age-dependent Tresp cell proliferation to normal levels, but simultaneously inhibited Treg cell proliferation below normal levels., Conclusions: Our data reveal that the currently used immunosuppressive therapy has a favorable effect on the differentiation and proliferation of Tresps but has a rather unfavorable effect on the proliferation of Tregs. Newer substances with more specific effects on the immune system would be desirable.

Diminishing potential to replace damaged tissues is a hallmark for ageing of somatic stem cells, but the mechanisms remain elusive. Here, we present proteome-wide atlases of age-associated alterations in human haematopoietic stem and progenitor cells (HPCs) and five other cell populations that constitute the bone marrow niche. For each, the abundance of a large fraction of the ~12,000 proteins identified is assessed in 59 human subjects from different ages. As the HPCs become older, pathways in central carbon metabolism exhibit features reminiscent of the Warburg effect, where glycolytic intermediates are rerouted towards anabolism. Simultaneously, altered abundance of early regulators of HPC differentiation reveals a reduced functionality and a bias towards myeloid differentiation. Ageing causes alterations in the bone marrow niche too, and diminishes the functionality of the pathways involved in HPC homing. The data represent a valuable resource for further analyses, and for validation of knowledge gained from animal models.

To understand the precise disease driving mechanisms in acute myeloid leukemia (AML), comparison of patient matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. In this analysis, we have examined the value of aldehyde dehydrogenase (ALDH) activity in combination with CD34 expression for the separation of HSC from LSC in 104 patients with de novo AML. The majority of AML patients (80 out of 104) had low percentages of cells with high ALDH activity (ALDH(+) cells; <1.9%; ALDH-rare AML), whereas 24 patients had relatively numerous ALDH(+) cells (≥1.9%; ALDH-numerous AML). In patients with ALDH-rare AML, normal HSC could be separated by their CD34(+) ALDH(+) phenotype, whereas LSC were exclusively detected among CD34(+) ALDH(-) cells. For patients with ALDH-numerous AML, the CD34(+) ALDH(+) subset consisted mainly of LSC and separation from HSC was not feasible. Functional analyses further showed that ALDH(+) cells from ALDH-numerous AML were quiescent, refractory to ARA-C treatment and capable of leukemic engraftment in a xenogenic mouse transplantation model. Clinically, resistance to chemotherapy and poor long-term outcome were also characteristic for patients with ALDH-numerous AML providing an additional risk-stratification tool. The difference in spectrum and relevance of ALDH activity in the putative LSC populations demonstrates, in addition to phenotypic and genetic, also functional heterogeneity of leukemic cells and suggests divergent roles for ALDH activity in normal HSC versus LSC. By acknowledging these differences our study provides a new and useful tool for prospective identification of AML cases in which separation of HSC from LSC is possible., (© 2014 UICC.)

Proteoglycans are often overexpressed in tumors and can be found on several normal and neoplastic stem cells. In this study, we analyzed in-depth the role of CSPG4 in head and neck squamous cell carcinomas (HNSCC). Analysis of CSPG4 in a homogeneous study sample of HPV-negative stage IVa HNSCCs revealed overexpression of protein and mRNA levels in a subgroup of HNSCC tumors and a significant association of high CSPG4 protein levels with poor survival. This could be validated in three publicly available microarray datasets. As a potential cause for upregulated CSPG4 expression, we identified DNA hypomethylation in a CpG-island of the promoter region. Accordingly, we found an inverse correlation of methylation and patient outcome. Finally, CSPG4 re-expression was achieved by demethylating treatment of highly methylated HNSCC cell lines establishing a direct link between methylation and CSPG4 expression. In conclusion, we identified CSPG4 as a novel biomarker in HNSCC on several biological levels and established a causative link between DNA methylation and CSPG4 protein and mRNA expression., (© 2014 UICC.)

Cancer cells with enhanced self-renewal capacity influence tumour growth in glioblastoma. So far, a variety of surrogate markers have been proposed to enrich these cells, emphasizing the need to devise new characterization methods. Here, we screen a large panel of glioblastoma cultures (n = 21) cultivated under stem cell-permissive conditions and identify several cell lines with enhanced self-renewal capacity. These cell lines are capable of matrix-independent growth and form fast-growing, orthotopic tumours in mice. Employing isolation, re-plating, and label-retention techniques, we show that self-renewal potential of individual cells is partitioned asymmetrically between daughter cells in a robust and cell line-specific fashion. This yields populations of fast- and slow-cycling cells, which differ in the expression of cell cycle-associated transcripts. Intriguingly, fast-growing cells keep their slow-cycling counterparts in a reversible state of quiescence associated with high chemoresistance. Our results suggest that two different subpopulations of tumour cells contribute to aberrant growth and tumour recurrence after therapy in glioblastoma., (Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Mounting evidences indicate that leukemic cells in patients with acute myeloid leukemia (AML) are derived from leukemia stem cells (LSC). In analogy to normal hematopoietic stem cells (HSC), LSC remain mostly dormant and are hence resistant to conventional chemotherapy. Residual, physiological HSC exist alongside with LSC, with heterogeneous dominance of LSC over HSC in individual patients. We have devised a flow cytometric method for the identification and separation of these two stem cell populations based on surface antigen markers such as CD34, CD38, lineage aberrant markers, and aldehyde dehydrogenase (ALDH) enzyme activity.

The biological significance of CD133 in glioblastoma is controversial. Above all, there is disagreement concerning the proper approach, the appropriate (cell) model and the suitable microenvironment to study this molecule, often leading to inconsistent experimental results among studies. In consideration of a primary need to dissect and to understand the CD133 phenotype in glioblastoma we performed a comprehensive analysis of CD133 expression and regulation in a large set of glioblastoma cell lines (n = 20) as well as in tumor xenografts. Our analysis considered alternatively spliced mRNA transcripts, different protein epitopes as well as varying sub-cellular localizations of CD133 and explored its regulation under pertinent micro-environmental conditions. CD133 mRNA and CD133 protein could be detected in all relevant types of glioblastoma cell lines. In addition, we detected frequent intracellular CD133 protein accumulations located to the ER and/or Golgi apparatus but seemingly unrelated to particular CD133 splice variants or protein epitopes. In contrast, membrane-bound expression of CD133 was restricted to tumor cells bearing the extracellular CD133 epitope AC133. Only in these cells, differentiation and oxygen levels clearly impacted on AC133 expression and to some extent also influenced CD133 mRNA and protein expression. Most importantly, however, modulation of AC133 levels could occur independently of changes in CD133 mRNA transcription, CD133 protein translation, protein retention or protein shedding. Our results suggest that the AC133 epitope, rather than CD133 mRNA or protein, mirrors malignancy-related tumor traits such as tumor differentiation and local oxygen tension levels, and thus corroborate its role as a biologically relevant cancer marker., (Copyright © 2011 Wiley‐Liss, Inc.)

DNA methylation plays an important role in the self-renewal of hematopoietic stem cells and in the commitment to the lymphoid or myeloid lineages. Using purified CD34⁺ hematopoietic progenitor cells and differentiated myeloid cell populations from the same human samples, we obtained detailed methylation profiles at distinct stages of hematopoiesis. We identified a defined set of differentiation-related genes that are methylated in CD34⁺ hematopoietic progenitor cells but show pronounced DNA hypomethylation in monocytes and in granulocytes. In addition, by comparing hematopoietic progenitor cells from umbilical cord blood to hematopoietic progenitor cells from peripheral blood of adult donors we were also able to analyze age-related methylation changes in CD34⁺ cells. Interestingly, the methylation changes observed in older progenitor cells showed a bimodal pattern with hypomethylation of differentiation-associated genes and de novo methylation events resembling epigenetic mutations. Our results thus provide detailed insight into the methylation dynamics during differentiation and suggest that epigenetic changes contribute to hematopoietic progenitor cell aging.

Background: The development of distant metastasis is associated with poor outcome in patients with colorectal cancer (CRC). The stromal cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) have pivotal roles in the chemotaxis of migrating tumor cells during metastasis. Thus, hampering the SDF-1/CXCR4 cross-talk is a promising strategy to suppress metastasis., Methods: We investigated the invasive behavior of the lentivirally CXCR4 overexpressing CRC cell lines SW480, SW620 and RKO in chemotaxis and invasion assays toward an SDF-1α gradient. Low endogenous CXCR4 expression levels were determined by quantitative realtime polymerase chain reaction (PCR) and fluorescence-activated cell sorting (FACS) analyses., Results: A lentiviral CXCR4 overexpression and knockdown model was established in these CRC cells. In transwell migration assays, CXCR4 overexpression favored chemotaxis and invasion of cells in all 3 lines depending on an SDF-1α gradient (p < 0.001 vs. untransduced cells). Functional CXCR4 knockdown using lentiviral short hairpin RNA (shRNA) vectors significantly decreased the migration behavior in CRC cell lines (p < 0.001), confirming a CXCR4-specific effect. Pharmacologic inhibition of the SDF-1α/CXCR4 interaction by the bicyclam Plerixafor(TM) at 100 μM significantly abrogated CXCR4-dependent migration and invasion through Matrigel(TM) (SW480, SW620, RKO; p < 0.05)., Conclusion: Our results indicate that a CXCR4-antagonistic therapy might prevent tumor cell dissemination and metastasis in CRC patients, consequently improving survival., (2011 S. Karger AG, Basel.)

Mesenchymal stromal cells (MSC) have been suggested to provide a suitable cellular environment for in vitro expansion of haematopoietic stem and progenitor cells (HPC) from umbilical cord blood. In this study, we have simultaneously analysed the cell division history and immunophenotypic differentiation of HPC by using cell division tracking with carboxyfluorescein diacetate N-succinimidyl ester (CFSE). Co-culture with MSC greatly enhanced proliferation of human HPC, especially of the more primitive CD34(+)CD38(-) fraction. Without co-culture CD34 and CD133 expressions decreased after several cell divisions, whereas CD38 expression was up-regulated after some cell divisions and then diminished in fast proliferating cells. Co-culture with MSC maintained a primitive immunophenotype (CD34(+), CD133(+) and CD38(-)) for more population doublings, whereas up-regulation of differentiation markers (CD13, CD45 and CD56) in HPC was delayed to higher numbers of cell divisions. Especially MSC of early cell passages maintained CD34 expression in HPC over more cell divisions, whereas MSC of higher passages further enhanced their proliferation rate. Inhibition of mitogen-activated protein kinase 1 (MAPK1) impaired proliferation and differentiation of HPC, but not maintenance of long-term culture initiating cells. siRNA knockdown of N-cadherin and VCAM1 in feeder layer cells increased the fraction of slow dividing HPC, whereas knockdown of integrin beta 1 (ITGB1) and CD44 impaired their differentiation. In conclusion, MSC support proliferation as well as self-renewal of HPC with primitive immunophenotype. The use of early passages of MSC and genetic manipulation of proteins involved in HPC-MSC interaction might further enhance cord blood expansion on MSC.

B cells are well-known mediators of humoral immunity and serve as costimulators in the generation of T cell-mediated responses. In several mouse models, however, it was observed that B cells can also down-regulate immune reactions, suggesting a dual role for B cells. Due to this discrepancy and so far limited data, we directly tested the effects of primary human B cells on activated CD4(+) T helper cells in vitro. We found that under optimal costimulation large, activated CD25(+) B cells but not small CD25(-) B cells induced temporary T-cell anergy, determined by cell division arrest and down-regulation of cytokine production. In addition, large CD25(+) B cells directly induced CD95-independent apoptosis in a subpopulation of activated T cells. Suppression required direct B-T-cell contact and was not transferable from T to T cell, excluding potential involvement of regulatory T cells. Moreover, inhibitory effects involved an IL-2-dependent mechanism, since decreasing concentrations of IL-2 led to a shift from inhibitory toward costimulatory effects triggered by B cells. We conclude that activated CD25(+) B cells are able to costimulate or down-regulate T-cell responses, depending on activation status and environmental conditions that might also influence their pathophysiological impact.

Objective: CD4+,CD25high regulatory T (Treg) cells play a crucial role in the maintenance of self tolerance and prevention of organ-specific autoimmunity. The presence of many in vivo-preactivated CD4+,CD25++ T cells in patients with systemic lupus erythematosus (SLE) poses a difficulty in discriminating CD25++ activated T cells from CD25high Treg cells. To overcome this problem, we analyzed the phenotype and function of CD4+,CD25high,CD127(-/low) natural Treg (nTreg) cells isolated from the peripheral blood of patients with SLE., Methods: CD4+,CD25high,CD127(-/low) nTreg cells and CD4+,CD25- responder T (Tresp) cells from patients with SLE and normal donors were separated by fluorescence-activated cell sorting. Cell proliferation was quantified by 3H-thymidine incorporation, and immunophenotyping of the cells was done using FACScan., Results: Comparable percentages of CD4+,CD25high,FoxP3+ T cells were observed in patients with SLE and normal donors. Proliferation of SLE nTreg cells sorted into the subset CD4+,CD25high,CD127(-/low) was significantly decreased compared with that of SLE nTreg cells sorted into the subset CD4+,CD25high (mean +/- SEM 2,223 +/- 351 counts per minute versus 9,104 +/- 1,720 cpm, respectively), while in normal donors, these values were 802 +/- 177 cpm and 2,028 +/- 548 cpm, respectively, confirming that effector cell contamination was reduced. Notably, the suppressive activity of nTreg cells was intact in all groups. However, CD4+,CD25- Tresp cells isolated from patients with active SLE were significantly less sensitive than those from patients with inactive SLE to the suppressive function of autologous or normal donor CD4+,CD25high,CD127(-/low) nTreg cells. Furthermore, a significant inverse correlation was observed between the extent of T cell regulation in suppressor assays and the level of lupus disease activity., Conclusion: This study is the first to show that, in human SLE, impaired sensitivity of Tresp cells to the suppressive effects of a comparably functional, highly purified nTreg cell population leads to a defective suppression of T cell proliferation in active SLE. Studies aiming to define the mechanisms leading to Tresp cell resistance might help in the development of highly specific, alternative immunotherapeutic tools for the control of systemic autoimmune diseases such as SLE.

Objective: Studies in animal models have indicated that hematopoietic progenitor cells (HPC) migrate and home to the central nervous system and might acquire neural features under specific circumstances. The interaction between HPC and the neural environment and the functional effect on hematopoiesis have not yet been defined., Methods: CD34(+)133(+) cells from mobilized peripheral blood were cocultured with primary murine neurons or astrocytes. Chemotaxis and adhesive interactions were studied by applying beta(1)- and beta(2)-integrin function-blocking anibodies. The impact of neural feeder layers on integrin expression of HPC and the presence of appropriate adhesion ligands on neural cells were determined by immunostaining and flow cytometry. The hematopoietic long-term fate was monitored by time-lapse microscopy of individual cell-division history followed by long-term culture-initiating cell (LTC-IC) and colony-forming cell (CFC) assays. Neural differentiation was assessed by immunostaining against specific neuronal and glial antigens., Results: The 23.0% +/- 4.9% of HPC showed stromal cell-derived factor-1-induced migration toward neural cells, and 20.2% +/- 1.6% displayed firm beta(1)-integrin-mediated adhesion to astrocytes. The latter expressed appropriate adhesion ligands, stabilized beta(1)-integrin expression, and increased beta(2)-integrin expression of HPC. Neural differentiation of HPC could not be identified but astrocytes were able to induce limited self-renewing cell divisions of HPC and thus maintain 25.8% +/- 3.4% of the initial LTC-IC and 80.7% +/- 1.9% of the initial CFC., Conclusion: Human HPC are able to interact with neural cells and interaction maintains, albeit to a limited extent, the self-renewal capability of HPC.