Intratumoral, Injection of Adenoviral Flt3 Ligand Has Therapeutic Activity in Association with Increased Intratumoral Levels of T Cells but Not Dendritic Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5280-5280
Author(s):  
James E. Talmadge ◽  
Scott G. Kurz ◽  
Randy R. Fields ◽  
Devendra K. Agrawal ◽  
Rakesh K. Singh ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Fold Increase ◽  
Mammary Tumors ◽  
Intratumoral Injection ◽  
Lymphoid Cells ◽  
Mammary Adenocarcinoma ◽  
Therapeutic Activity ◽  
Primary Tumors ◽  
Hematopoietic Stem

Abstract Fms-like tyrosine kinase 3 ligand (Flt3L) is a growth factor for dendritic cells (DCs), hematopoietic stem cells, and natural killer (NK) cells in vivo. Ten daily injections of Flt3L, systemically expands DCs, whereas a single injection of an adenovirus vector with the Flt3L transgene (Adv-Flt3L) significantly expands DCs as soon as 2 days following intravenous (iv) injection. The increases in DC numbers, following iv injection, remains at near peak levels from day 6 through day 16. Maximal DC expansion occurs following the iv injection of 1011 virus particles, such that eight days following the iv injection of Adv-Flt3L it induced an 8 fold increase in splenic CD11c+CD11b−CD8a+ cells (immune augmenting DCs), a 6 fold increase in splenic CD11c+B220+ plasmacytoid DCs, and a 9 fold increase in splenic CD11c+CD11b+ cells (immunosuppressive DCs). The increase in DC numbers and cellularity returns to near normal levels by 22 days following iv injection of Adv-Flt3L. Despite the 2–3 fold increase in spleen cellularity and significant expansion of DCs in the spleen, blood and lungs by the iv injection of Adv-Flt3L, this route of administration has minimal to no therapeutic activity for orthrotopic mammary tumors, even when therapy is initiated against small primary tumors. In contrast, the intratumoral injection of Adv-Flt3L has significant therapeutic activity against orthrotopic mammary tumors including mice in which therapy was initiated against large tumors. Unexpectedly, no significant increase in DC infiltration of the tumor was observed following iv or intratumoral Adv-Flt3L administration. In contrast, the number of tumor infiltrating CD4+ and CD8+ cells was increased following intratumoral but not iv Adv-Flt3L administration, suggesting an association with therapeutic activity. Adv-Flt3L injection also induced DC expansion in the spleen, PB and lungs in cl-66, mammary adenocarcinoma-bearing mice. Injection of adv-Flt3L (iv or intratumoral) also stimulated the expansion of T-cells (both CD4+ and CD8+) and a type 1 T cell response as measured by qRT-PCR. Further, the therapeutic activity and increased mitogenic responses by splenic lymphoid cells to Con-A and IL-2 was not depressed in mice bearing cl-66 tumors and the effect of Adv-Flt3L, while partially associated with the Adv vector, was significantly higher in mice injected with Adv-Flt3L.

Diversity, localization and (patho)physiology of mature lymphocyte populations in the bone marrow

Blood ◽  
2021 ◽  
Author(s):  
Christian M. Schürch ◽  
Chiara Caraccio ◽  
Martijn A. Nolte
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Plasma Cells ◽  
Cell Types ◽  
Lymphoid Organ ◽  
Lymphoid Cells ◽  
Term Survival ◽  
Hematopoietic Stem ◽  
Mature Lymphocyte ◽  
Lymphocyte Populations

The bone marrow (BM) is responsible for generating and maintaining lifelong output of blood and immune cells. Besides its key hematopoietic function, the BM acts as an important lymphoid organ, hosting a large variety of mature lymphocyte populations, including B-cells, T-cells, NK(T)-cells and innate lymphoid cells (ILCs). Many of these cell types are thought to only transiently visit the BM, but for others, like plasma cells and memory T-cells, the BM provides supportive niches that promote their long-term survival. Interestingly, accumulating evidence points towards an important role for mature lymphocytes in the regulation of hematopoietic stem cells (HSCs) and hematopoiesis in health and disease. In this review, we describe the diversity, migration, localization and function of mature lymphocyte populations in murine and human BM, focusing on their role in immunity and hematopoiesis. We also address how various BM lymphocyte subsets contribute to the development of aplastic anemia and immune thrombocytopenia, illustrating the complexity of these BM disorders, but also the underlying similarities and differences in their disease pathophysiology. Finally, we summarize the interactions between mature lymphocytes and BM resident cells in HSC transplantation and graft-versus-host disease. A better understanding of the mechanisms by which mature lymphocyte populations regulate BM function will likely improve future therapies for patients with benign and malignant hematological disorders.

scholarly journals Transcription factor Etv6 regulates functional differentiation of cross-presenting classical dendritic cells

2018 ◽  
Vol 215 (9) ◽  
pp. 2265-2278 ◽  
Author(s):  
Colleen M. Lau ◽  
Ioanna Tiniakou ◽  
Oriana A. Perez ◽  
Margaret E. Kirkling ◽  
George S. Yap ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Transcription Factor ◽  
T Cell ◽  
Cd8 T Cells ◽  
Functional Differentiation ◽  
Specific Gene ◽  
Hematopoietic Stem

An IRF8-dependent subset of conventional dendritic cells (cDCs), termed cDC1, effectively cross-primes CD8+ T cells and facilitates tumor-specific T cell responses. Etv6 is an ETS family transcription factor that controls hematopoietic stem and progenitor cell (HSPC) function and thrombopoiesis. We report that like HSPCs, cDCs express Etv6, but not its antagonist, ETS1, whereas interferon-producing plasmacytoid dendritic cells (pDCs) express both factors. Deletion of Etv6 in the bone marrow impaired the generation of cDC1-like cells in vitro and abolished the expression of signature marker CD8α on cDC1 in vivo. Moreover, Etv6-deficient primary cDC1 showed a partial reduction of cDC-specific and cDC1-specific gene expression and chromatin signatures and an aberrant up-regulation of pDC-specific signatures. Accordingly, DC-specific Etv6 deletion impaired CD8+ T cell cross-priming and the generation of tumor antigen–specific CD8+ T cells. Thus, Etv6 optimizes the resolution of cDC1 and pDC expression programs and the functional fitness of cDC1, thereby facilitating T cell cross-priming and tumor-specific responses.

Effects of B7.2−/− Mature Dendritic Cells on Tolerance Induction to Alloantigens in Fetal Mice Following In Utero Transplantation with Lineage Depleted Bone Marrow.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2134-2134
Author(s):  
Swati Bhattacharyya ◽  
Morton J. Cowan
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Dendritic Cells ◽  
Stem Cell ◽  
In Utero ◽  
Relative Role ◽  
Skin Grafts ◽  
Hematopoietic Stem ◽  
Fetal Mice

Abstract In utero hematopoietic stem cell transplantation (IUT) has the potential to cure a variety of marrow stem cell defects without using marrow ablative therapy. However IUT for diseases other than SCID has been unsuccessful. To better understand the barriers to successful IUT we wanted to define the role of the B7.1/B7.2 co-stimulatory molecules in inducing tolerance to allogeneic donor bone marrow cells in the fetal murine recipient. We studied the relative role of B7.1 and B7.2 expression on dendritic cells (DC) on engraftment and in generating donor specific tolerance in fetal mice. Mature DC (mDC) from B7.1−/− or B7.2−/− donors and wild type (wt) lineage depleted (lin−) C57Bl/6 (B6) bone marrow (BM) were injected into gestational day (GD) 14 Balb/c fetuses. Recipients of lin− wt BM and B7.1−/− mDC had a significantly lower survival (47.4%, p<0.01) associated with mild-moderate GvHD compared to the recipients of B7.2−/− mDC and lin− BM (82.3%) where none developed GvHD. Engraftment results in blood at 6 weeks post IUT showed, B7.1−/− recipients had multilineage engraftment (4.7±0.8% T cells and 5.7± 1.1% granulocytes) in their blood, but by 12 weeks, only donor CD3+ (predominantly CD8+) cells (2.1±1.3%) were present. The percent H2Kb+ (donor) T cells (predominantly CD4+) in the blood of recipients of lin− wt BM and B7.2−/− was 11.8±8.5% at 6 weeks p<0.001 and 6.5±2.5% at 12 weeks, p=0.006. The circulating donor CD4+ cells were Th2 (CD4+CD25−IL4+IL10+) and Treg (CD4+CD25+IL4−IL10−). Both fractions inhibited the T cell proliferative response in the MLR. Long term engraftment in thymic tissues was found in the tolerant recipients of lin− wt BM and B7.2−/− mDC (13.4±8.3% donor CD3+ T cells). We also found prolonged (rejection by day 36) acceptance of donor skin grafts in 7 of 12 recipients of B7.2−/− mDC and 2 of 5 recipients of B7.2−/− mDC and lin−BM. All third party C3H grafts were rejected by day 14 and 80% of the Balb/c (self) skin grafts were permanently accepted. We hypothesized that tolerized animals would behave similarly to recipients of megadoses of syngeneic BM with an increase in multilineage engraftment. We injected a total of 200x106 male wt B6 lin− BM cells over 5 days into adult IUT recipients of B7.1−/− or B7.2−/− mDC ± lin− wt BM and wt age-matched allogeneic and syngeneic (female) controls. Mice that had received B7.2−/− mDC + lin− BM in utero showed multi-lineage engraftment in the blood. In contrast, the in utero recipients of B7.1−/− mDC + lin− BM showed no significant engraftment (p<0.05). In conclusion, donor DC costimulatory molecules significantly affect survival, engraftment and GvHD; and these responses to B7.2−/− mDC and lin− BM appear to be mediated by both Th2 and Treg donor cells.

Human CD34+Cells Mobilized by AMD3100 Demonstrate Enhanced NOD/SCID Repopulating Function Compared to CD34+ Cells Mobilized by Granulocyte Colony Stimulating Factor.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1962-1962 ◽  
Author(s):  
David A. Hess ◽  
Louisa Wirthlin ◽  
Timothy P. Craft ◽  
Jesper Bonde ◽  
Ryan W. Lahey ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Dna Sequences ◽  
Peripheral Blood ◽  
Paired Comparison ◽  
Fold Increase ◽  
Human Cells ◽  
Lymphoid Cells ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Interactions between stromal derived factor-1 (SDF-1 or CXCL12), and its receptor CXCR4 regulate hematopoietic stem and progenitor cell retention in the bone marrow. AMD3100, a bicyclam molecule that selectively blocks the interaction between CXCL12 and CXCR4, has recently been used in clinical trials to rapidly mobilize hematopoietic progenitor cells. However, the functional properties of human stem and progenitor cells mobilized with this agent are not well characterized. Here, we directly compared the NOD/SCID repopulating function of CD34+ cells rapidly mobilized (4 hours) by AMD3100 versus CD34+ cells mobilized after 5 days of G-CSF treatment. A total of 7 HLA-matched sibling donors were leukapheresed after a single injection of 240ug/kg AMD3100. After 1 week of drug clearance, the same donor was mobilized with G-CSF, allowing a paired comparison of the repopulating function of cells mobilized by the two agents. Total CD34+ cells mobilized by AMD3100 treatment averaged 1.2±0.4x106 CD34+ cells/kg (range 0.4–2.1x106 CD34+ cells/kg), as compared to G-CSF treatment at 3.2±0.9x106 CD34+ cells/kg (range 1.7–5.7 x106 CD34+ cells/kg). Leukapheresis total mononuclear cell (MNC) fraction or purified CD34+ cells (>90% purity), were isolated and transplanted into sublethally irradiated NOD/SCID mice at varying doses. BM, spleen, and peripheral blood of mice were harvested 7–8 weeks post-transplantation and analyzed by flow cytometry for the presence or absence of engrafting human cells. Low frequency human engraftment events (<0.2% human cells) were confirmed by PCR for P17H8 alpha-satellite human DNA sequences. Injection of 1–40x106 MNC or 0.5–5x105 CD34+ cells produced consistent human engraftment and allowed limiting dilution analysis using Poisson statistics to be performed on paired samples of AMD3100 and G-CSF leukapheresis products from 3 individual patients. The calculated frequencies of NOD/SCID repopulating cells (SRC) were 1 SRC in 11.5x106 AMD3100-mobilized MNC (n=50) compared to 1 SRC in 44.8x106 G-CSF-mobilized MNC (n=55). For purified CD34+ populations, the overall frequency of repopulating cells was 1 SRC in 1.0x105 AMD3100-mobilized CDC34+ cells (n=53) compared to 1 SRC in 3.1x105 G-CSF-mobilized CD34+ cells (n=45). These data correspond to a 3–4-fold increase in overall repopulating function demonstrated by AMD3100 mobilized cells. Multilineage hematopoietic differentiation of transplanted CD34+ cells was similar for AMD3100 and G-CSF-mobilized CD34+ cells, with equivalent production of myelo-monocytic cells (CD33+CD14+), immature B-lymphoid cells (CD19+CD20+), and primitive repopulating (CD34+CD133+CD38−) cells 7–8 weeks post-transplantation. These studies indicate that human AMD3100-mobilized MNC and purified CD34+ cells possess enhanced repopulating capacity, as compared to G-CSF mobilized counterparts from the same donor. Thus, AMD3100 mobilized peripheral blood represents a rapidly obtained and highly functional source of repopulating hematopoietic stem cells for clinical transplantation procedures.

Host Plasmacytoid or Conventional Dendritic Cells Alone Are Sufficient To Initiate Graft-Versus-Host Disease.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2164-2164
Author(s):  
Motoko Koyama ◽  
Daigo Hashimoto ◽  
Kazutoshi Aoyama ◽  
Ken-ichi Matsuoka ◽  
Kennosuke Karube ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
B Cells ◽  
Mhc Class Ii ◽  
Graft Versus Host Disease ◽  
Class Ii ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Host Disease ◽  
Graft Versus Host

Abstract Graft-versus-host disease (GVHD) is a major complication after allogeneic hematopoietic stem cell transplantation. Alloantigen expression on host dendritic cells (DCs) is critical to initiate GVHD. DCs can be divided into two main subpopulations; conventional DCs (cDCs) and plasmacytoid DCs (pDCs), however, the contribution of each DC subset to elicit GVHD remains unclear. We examined the ability of cDCs and pDCs to initiate GVHD. pDCs, cDCs and B cells were isolated from C57BL/6 (B6: H–2b) mice treated with Flt3 ligand in order to expand DCs. pDCs were enriched from bone marrow by depleting CD3+, CD19+, CD11b+, and CD49b+ cells, followed by a FACS sorting of CD11cint B220+ cells. cDCs and B cells were sorted from splenocytes as CD11chi B220− cells and CD11c− B220+ cells, respectively. Isolated pDCs showed plasmacytoid morphology, produced IFN-α in response to CpG oligonucleotide. Although pDCs stimulated allogeneic T cells far less potently than cDCs, stimulation with CpG enhanced their allostimulatory capacity as potent as cDCs. We compared the ability of each DC subset to initiate GVHD by an add-back study of MHC class II-expressing DCs into MHC class II-deficient (II−/−) mice that were resistant to CD4-dependent GVHD. Lethally irradiated II−/− B6 mice were injected with 2 × 106 pDCs, cDCs or B cells from wild-type (II+/+) B6 mice on day -1 and injected with 2 × 106 CD4+ T cell from BALB/c (H–2d) mice on day 0. A flow cytometric analysis of the mesenteric lymph nodes on day +5 demonstrated significantly greater expansion of donor CD4+ T cells in recipients of pDCs or cDCs than those of B cells (Table). While injection of B cells did not cause any sign of GVHD, injection of pDCs or cDCs alone was sufficient to produce clinical and pathological GVHD (Table), thus breaking GVHD resistance of II−/− mice. We next examined the ability of pDCs to induce CD8-dependent GVHD in MHC-matched transplant using mice deficient in functional MHC class I expression (β2m−/−). Again, injection of pDCs or cDCs alone was sufficient to cause expansion of donor CD8+ T cells (p&lt;0.05). We next asked whether signaling through Toll-like receptors (TLRs) could be required for pDCs to initiate GVHD. However, injection of pDCs isolated from MyD88/TRIF-double deficient mice was able to initiate GVHD as potent as wild-type pDCs, thus demonstrating that pDCs initiate GVHD in a TLR signaling independent manner. These results provide important information for developing strategies aimed at inactivating host DCs to prevent GVHD. Impact of each APC subpopulation on GVHD APC Donor CD4 expansion (×103±SE) Clinical GVHD score (mean±SE) Pathological GVHD score (mean±SE) *p&lt;0.05 compared with B cells B cell 0.1 ± 0.0 2.1 ± 0.2 2.1 ± 0.2 pDC 5.3 ± 2.4* 4.3 ± 0.3* 7.4 ± 0.5* cDC 9.7 ± 3.8 * 3.8 ± 0.5 * 7.2 ± 0.7*

Increased Expression Of IL-15 Promotes Cutaneous T-Cell Lymphomagenesis Via The Upregulation Of Histone Deacetylases: Evidence For Successful Preclinical Targeting

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1826-1826
Author(s):  
Anjali Mishra ◽  
Krista M.D. La Perle ◽  
Laura Sullivan ◽  
Gregory H Sams ◽  
Douglas P Curphey ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Histone Deacetylases ◽  
Flow Cytometric Analysis ◽  
Fold Increase ◽  
Lymphoid Cells ◽  
Cutaneous Lymphoma ◽  
Normal Donor

Abstract Cutaneous lymphoma is a heterogeneous group of neoplasms of skin-homing malignant lymphocytes. Cutaneous T-cell Lymphoma (CTCL) represents 70-80% of all cutaneous lymphoma and its pathogenesis is largely unknown. Previous studies have shown that interleukin (IL)-15 is a potent stimulant and growth factor for CTCL cells in vitro. In order to investigate the intrinsic levels of IL-15 in CTCL patients, malignant CD4+ T-cells were analyzed for expression of IL-15. Relative quantitation of IL-15 transcript in patient vs normal donor (ND) CD4+ cells showed overexpression of IL-15 in patients (fold increase mean ± SD = 5.36 ± 4.39, N=13, P<0.001). Increase in IL-15 transcript was directly proportional to disease severity in patients i.e. fold increase mean ± SD in IL-15 in Stage I =3.28 ± 1.42, N=3 each, P=0.0047 vs. Stage III patients = 7.42 ± 1.30, N=3 each, P=0.0073. Further, cutaneous lesions in patients stained positive for IL-15 protein in atypical lymphoid cells and Pautrier's microabscess. We next investigated the role of IL-15 in CTCL development using IL-15 transgenic (tg) mice. Within 4-6 weeks of birth, IL-15 tg mice developed extensive patch/plaque skin lesions, progressive alopecia, and severe pruritus. Adult IL-15 tg mice developed extensive involvement with cutaneous lymphoma that was fatal in 100% of the mice (P=0.0003). Antibodies staining revealed that CD4+ skin resident T-cells in IL-15 tg mice were CD3+CD62L-CD44hiCCR4+CLA+. Flow cytometric analysis of single cell suspension of skin showed ∼25-fold increase in CD3+ T-cells in IL-15 tg compared to WT controls (Mean ± SD of absolute number of cells= 3.80 ± 6.97, N=14 vs. 0.15 ± 0.26, N=8 respectively, P<0.001). Lymphoma cells from IL-15 tg mouse skin engrafted and mimicked the primary disease in immune deficient SCID mice upon adoptive transfer. CD4+ T-cells from CTCL patients showed increased histone deacetylases (HDAC) 1, HDAC2 and HDAC6 transcripts over ND CD4+ T-cells and immunoblot analysis of ND CD4+ T-cells exposed to 100ng/ml IL-15 showed upregulation of HDAC1, HDAC2 and HDAC6 ex vivo. IL-15 stimulation of ND CD4+ T-cells resulted in loss of expression of the downstream HDAC1/2 target tumor suppressor, p21 in vitro, and knock down of HDAC6 in IL-15 stimulated ND CD4+ T-cells inhibited their migration in vitro; suggesting that IL-15 mediated upregulation of HDAC6 is critical for T-cell migration. Considering these observations, we used specific novel HDAC inhibitors (HDACi) to target HDAC1/2 (JQ12) and/or HDAC6 (WT161) in IL-15 tg mice to determine if we could prevent CTCL in vivo. IL-15 tg mice were treated with 50mg/kg of either or both the inhibitors, 5 days/week for 4 weeks (n=4 each). While placebo treated IL-15 tg mice progressively developed lesions during the course of treatment, IL-15 tg mice treated with JQ12 and/or WT161 showed no clinical signs of disease. This was further corroborated by histopathology analysis of skin sections from treated mice (Figure 1). Thus, our data suggest that inhibiting HDAC1, HDAC2 and/or HDAC6 pathways inhibits the development of CTCL in IL-15tg mice. In addition to the prevention study, we assessed the ability of a novel pan-HDACi, AR42, to treat active and progressive disease in our model. IL-15 tg mice with established CTCL were randomized to receive either AR42 or placebo feed (n=6 each) for 12 days. The IL-15 tg mice treated with AR42 showed remarkable improvement compared to the placebo mice whose disease progressed. Histopathology analysis of the AR42-treated IL-15 tg mice showed an impressive clearance of the CD3+ and CD4+ atypical lymphocytic infiltrate compared to placebo-treated mice (Figure 2). In summary we provide evidence that IL-15 has a causal role in the pathogenesis of CTCL; that IL-15 tg mice provide a novel model for studying disease pathogenesis and for evaluating potential therapies; that HDACi targeting specific HDACs may be effective in preventing CTCL and a novel pan-HDACi can reverse severe dermatologic disease in this CTCL model. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures: No relevant conflicts of interest to declare.

Serum Mir-29a Is Up-Regulated In Acute Graft Versus Host Disease (aGVHD) After Allogeneic Hematopoietic Stem Cell Transplantation (allo HSCT) and Activates Dendritic Cells (DCs)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4471-4471 ◽  
Author(s):  
Yvonne A Efebera ◽  
Parvathi Ranganathan ◽  
Xueyan Yu ◽  
Jessica Hofstetter ◽  
Sabrina L Garman ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Mirna Expression ◽  
Conditioning Regimen ◽  
Mouse Splenocytes ◽  
Hematopoietic Stem ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Serum Mirna ◽  
293 Cells

Background aGVHD is one of the most frequent and lethal complications after allo HSCT, underscoring the need to develop novel therapies. To achieve this goal, aGVHD mechanisms needs to be further elucidated. Recently it was reported that miRNAs are modulating aGVHD. In addition miRNAs are also present in the human serum and regulate immune responses. Here, we hypothesize that serum miRNAs expression is deregulated in aGVHD and could play a role in aGVHD pathogenesis. Methods To identify miRNAs associated with aGVHD we performed serum miRNA expression analysis using deep-sequencing from allo HSCT recipients samples at the time of clinical suspicion of aGVHD. Peripheral blood (PB) samples were collected weekly until day 100+ and at the time of clinical diagnosis of aGVHD from allo HSCT patients enrolled into OSU11002. After serum separation, total RNA was extracted using Trizol. Libraries were constructed using the small RNA profiling kit and sequenced on the Solid analyzer. A mouse model of aGVHD (B6 mice donor splenocytes and BM cells transplanted to lethally irradiated F1 recipients) was used to assess serum miRNA expression in animals with aGVHD. Results In this study we included 10 patients with aGVHD (bowel n=2; skin (n=5) and both skin and bowel aGVHD (n=3). Median age was 51.9, conditioning regimens were mainly non-myeloablative (n=9), with unrelated donors (n=9). PB samples from allo HSCT patients with no aGVHD and matched for age, disease, conditioning regimen, donor and timing of sample collection were obtained and used as controls. Sequence alignment was performed using miRBase. Normalization as reads per million was followed by quantiles. We compared miRNA expression between all patients with aGVHD (n=10) and controls (n=7) using class comparison (BRB). We found 7 miRNAs up-regulated (miR-146a, miR-323-b, miR-34c, miR-363, miR-4245, miR-29a, miR-181a* ) and 3 miRNAs down-regulated (miR-3168, miR-662, miR-550a) (Fold change (FC) >2, p<0.01). Since miR-146 and miR-29a were both involved in immune regulation we further validated these miRNAs by RT-PCR in the B6-F1 model of murine aGVHD. We found up-regulation of miR-146 FC 2, p<0.01 and miR-29a FC 4.9 p<0.01) in mice with aGVHD (n=6) with respect to controls (n=4). Next, we focus on miR-29a since our group found that this miR binds as ligands to TLR8. We hypothesized that serum miR-29a could bind to TLR8 of APCs activating NFkB and enhancing alloreactive responses during aGVHD. First, we examined whether extracellular miR-29a could activate dendritic cells (DCs). B6 splenocytes were stimulated with Dotap formulations (mimicking exosomes) of miR-29a. Negative controls included Dotap alone or Dotap-miR16 formulation. We found that CD69 expression measured by FACS is significantly elevated in CD11c+ DCs (34%), and CD8+ T cells (56%) populations treated with miR-29a compared to controls (p<0.01). CD86, a co-stimulatory molecule on DCs, was also significantly up-regulated after miR-29a stimulation (33%, p<0.01). To investigate whether T cells could be activated by the miR alone, independently of APCs, we isolated untouched resting T cells from mouse splenocytes suspension using the Pan-T cell isolation kit and stimulated them with Dotap-miR-29a, Dotap-miR-16 or Dotap alone. CD69 was not up-regulated under these culture conditions indicating that the activation of T cells was dependent on APCs activation. To further confirm that miR-29a could activate DCs, we isolated DCs from B6 mice using the pan DC isolation kit and repeated the above experiment. We found that miR-29a stimulation of DCs but not controls induced the up-regulation of both CD69 and CD86 (20%). Furthermore, miR-29a Dotap treatment of isolated DCs stimulated the release of TNFα in the supernatant (114.2±14.3 pg/ml vs. controls 26.98±2.09 pg/ml, p<0.01). We also performed coimmunoprecipitation assays for TLR8 in HEK-293 cells expressing GFP-TLR8 and treated with Dotap-miR-16, Dotap-miR-29a, or Dotap alone and determined miRNA levels by qRT-PCR. Only miR-29a expression was highly enriched (>50-fold). This binding leads to the activation of NFkB as measured by a NF-κB assay in TLR8–HEK-293 cells treated only with Dotap-miR-29a. Validation of these results using murine and human DCs are undergoing. Summary Altogether, our results indicate that serum miR-29a is up-regulated during aGVHD and activates DCs, likely by direct binding to TLR8 and inducing NFkB activation Disclosures: No relevant conflicts of interest to declare.

Single-Cell Immune Signatures in Patients with Chronic Phase Chronic Myeloid Leukemia (CML) Treated with Nilotinib: An ENEST1st Sub Study

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4022-4022
Author(s):  
Stein-Erik Gullaksen ◽  
Jørn Skavland ◽  
Krzysztof Warzocha ◽  
Sonia Gavasso ◽  
Vinko Tosevski ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Dendritic Cells ◽  
Regulatory T Cells ◽  
Cell Surface ◽  
Progenitor Cells ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Hematopoietic Stem ◽  
Additional Cell

Abstract Earlier evaluation of therapy effect in patients with CML would assist in optimal use of available tyrosine kinase inhibitors (TKI). Single cell analysis by mass cytometry has enabled the quantification of up to 46 antibody epitopes, making it ideally suited for exhaustive immunophenotyping of the haematological hierarchy, and evaluation of associated dynamic signal transduction events, in a clinical setting. By integrating time resolved single cell signalling data with clinical parameters, we searched for prognostic and efficacy-response mass cytometry biomarkers within a month of TKI therapy. We report data from experiments used to validate the custom panels of antibodies, highlighting the power of mass cytometry in the analysis of primary patient material obtained on clinical studies. Peripheral Blood (PB) samples were collected before, 3 hours, 7 days and 28 days, after start of nilotinib (300 mg BID) treatment in a subset of patients (n=55) enrolled in the ENEST1st trial. PB cells were stained with two panels of antibodies, allowing a comprehensive immunophenotyping of numerous cellular subsets, and also the evaluation of intracellular phosphorylation status of several epitopes. Moreover, using a straightforward barcoding scheme, the time-resolved samples from each individual patient were pooled after barcoding and stained with the antibody panels to minimize sample variation. In a pilot study, 7 and 10 cell subsets were identified in PB samples from 4 untreated healthy donors and 2 complete sets of 4 patients enrolled in this sub study, respectively. Furthermore, a robust signal was measured for pCrkL, pStat5, pStat3, pCreb, pAbl Y412 and pAbl Y245. The two sets of samples from study patients showed substantial changes in activation status over the course of therapy. Some changes, such as pStat3 alterations are only detectable in neutrophils and monocytes, while the activity of others i.e. pCreb was found to be ubiquitous. CD34+ cells indicated decreased phosphorylation of CrkL, Stat5, and Abl Y412/245. To increase the immunophenotyping resolution of the myeloid lineage, 3 additional cell surface markers were incorporated into the cell surface panel. In 1 healthy donor, and in diagnostic samples from three patients enrolled in this sub study, this allowed the identification of 13 cell subsets: CD3+, CD4+, and CD8+ T cells, regulatory T cells (Tregs), monocytes, dendritic cells (DCs), plasmacytoid dendritic cells (pDC's), neutrophils, basophils, B cells, hematopoietic stem cells (Lin- CD34+ CD38-) and progenitor cells (Lin- CD34+ CD38-) (Figure 1 A,B). With respect to the relative number of cells identified for each cell type, the three diagnosis samples differed from the single healthy control. In the patients, we observed an expansion of the granulocytic compartment, as well as the emergence of CD34+ progenitor and stem cells in the peripheral blood. In conclusion, the here presented developed assay is able to resolve most of the cell subpopulations found in the hematopoietic tree, and also robustly measure the activity of central signalling substrates known to be involved in CML pathogenesis. With the addition of new phospho-specific antibodies, the methodology may facilitate the detailed characterization of CML in an immunological context, and may shed new light on both the disease and therapeutic mechanism. Analysis of variation in signal responses and immune profile are now in progress in the subset of patients (n=55) in the ENEST1st trial. Figure 1. Manually annotated SPADE tree from healthy donor and patient (3581_0002). With the incorporation of additional cell surface markers, the protocol was able to identify 13 cellular subsets in healthy donors (A) and a typical CML patient (B): CD3+, CD4+, and CD8+ T cells, regulatory T cells (Tregs), monocytes, dendritic cells (DCs), plasmacytoid dendritic cells (pDC's), neutrophils, basophils, B cells, hematopoietic stem cells (Lin- CD34+ CD38-) and progenitor cells (Lin- CD34+ CD38-). Figure 1. Manually annotated SPADE tree from healthy donor and patient (3581_0002). With the incorporation of additional cell surface markers, the protocol was able to identify 13 cellular subsets in healthy donors (A) and a typical CML patient (B): CD3+, CD4+, and CD8+ T cells, regulatory T cells (Tregs), monocytes, dendritic cells (DCs), plasmacytoid dendritic cells (pDC's), neutrophils, basophils, B cells, hematopoietic stem cells (Lin- CD34+ CD38-) and progenitor cells (Lin- CD34+ CD38-). Disclosures Thaler: AOP Orphan: Research Funding. Lang:Celgene: Consultancy. Hjorth-Hansen:Bristol-Myers Squibb: Research Funding; Ariad: Honoraria; Novartis: Honoraria; Pfizer: Honoraria, Research Funding. Hellmann:Novartis: Consultancy, Other: funding of travel, accomodations or expenses, Research Funding, Speakers Bureau; BMS: Consultancy, Other: funding of travel, accomodations or expenses, Speakers Bureau. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding. Janssen:ARIAD: Consultancy; Bristol Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis: Research Funding. Porkka:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Ossenkoppele:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Mustjoki:Signe and Ane Gyllenberg Foundation: Research Funding; Finnish Cancer Institute: Research Funding; Sigrid Juselius Foundation: Research Funding; Pfizer: Honoraria, Research Funding; the Finnish Cancer Societies: Research Funding; Academy of Finland: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Gjertsen:Bergen University Hospital: Research Funding.

scholarly journals Exosomes Released From Human Bone Marrow–Derived Mesenchymal Stem Cell Attenuate Acute Graft-Versus-Host Disease After Allogeneic Hematopoietic Stem Cell Transplantation in Mice

2021 ◽  
Vol 9 ◽  
Author(s):  
Ke-Liang Li ◽  
Jin-Yan Li ◽  
Gui-Ling Xie ◽  
Xiao-Yan Ma
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Dendritic Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Clinical Score ◽  
Hematopoietic Stem ◽  
Graft Versus Host ◽  
Acute Graft

ObjectiveMesenchymal stromal cell–derived exosomes have been applied for the treatment of several immune diseases. This study aimed to explore the effect of human bone marrow–derived mesenchymal stem cell (hBMSC)–derived exosomes on acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT).MethodshBMSC were cultured, and the culture supernatants were then collected to prepare exosomes using total exosome isolation reagent from Invitrogen. Mouse aGVHD model was established by allogeneic cell transplantation and injected with hBMSC-derived exosomes (Msc-exo) via tail vein. Exosomes from human fibroblast (Fib-exo) were used as the treatment control. The effects of Msc-exo on dendritic cells, CD4+, and CD8+ T cells in aGVHD mice were analyzed through flow cytometry. The impact on inflammatory cytokines was tested by ELISA. Besides, the body weight, survival rate, and clinical score of treated mice were monitored.ResultsMsc-exo were successfully prepared. aGVHD mice injected with Msc-exo led to 7–8-fold increase of the CD8α+ conventional dendritic cells (cDCs) and CD11b+ cDCs compared with the controls. In addition, Msc-exo altered the T help and Treg subpopulation, and decreased the cytotoxicity and proliferation of cytotoxic T cells to favor inflammatory inhibition in aGVHD mice. Mice that received Msc-exo exhibited decreased weight loss and reduced aGVHD clinical score in a time-dependent manner as well as reduced lethality compared with Fib-exo treated or untreated control. Furthermore, the levels of IL-2, TNF-α, and IFN-γ were decreased, as well as the level of IL-10 was increased after Msc-exo treatment in vivo and in vitro.ConclusionhBMSC-derived exosomes could attenuate aGVHD damage and promote the survival of aGVHD mice by regulating the DC and T-cell subpopulation and function, and lead to inhibited inflammatory response in aGVHD mice.

scholarly journals P-selectin glycoprotein ligand-1 is broadly expressed in cells of myeloid, lymphoid, and dendritic lineage and in some nonhematopoietic cells

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 3010-3021 ◽  
Author(s):  
Z Laszik ◽  
PJ Jansen ◽  
RD Cummings ◽  
TF Tedder ◽  
RP McEver ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Myeloid Cells ◽  
Lymphoid Cells ◽  
Lymphoid Tissues ◽  
Follicular Dendritic Cells ◽  
Microvascular Endothelium ◽  
Segmented Neutrophil ◽  
Circulating Lymphocytes ◽  
Leukocyte Populations

P-selectin glycoprotein ligand-1 (PSGL-1) is a muclin-like glycoportein ligand for P- and E-selectin on myeloid cells and a subset of lymphocytes. We used flow cytometry and immunohistochemistry to examine expression of PSGL-1 on minor leukocyte populations, differentiating hematopoletic cells, and nonhematopoietic tissues using two monoclonal antibodies to distinct protein epitopes on PSGL-1. In the bone marrow, PSGL-1 was expressed on myeloid cells from the myeloblast stage to the segmented neutrophil, but was not detected on erythroblasts or megakaryocytes. All types of circulating myeloid cells expressed PSGL-1, and PSGL-1 was retained after extravasation of myetoid cells into tissues. PSGL-1 was also expressed on circulating dendritic cells, monocyte-derived dendritic cells, dendritic cells in lymphoid tissues and epidermis, and follicular dendritic cells. All types of lymphoid cells examined expressed PSGK-1, including immature and mature thymocytes, naive and memory T cells, gamma/delta T cells, netural killer cells, B cells and CD34+ progenitor cells. However, PSGL-1 levels were substantially lower on tonsillar lymphocytes than on circulating lymphocytes, suggesting that PSGL-1 expression is down regulated during or after entry of lymphocytes into secondary lymphoid tissue. Although PSGL-1 antigen was detected primarily on hamatopoietic cells, it was also present on time epithelium of the fallopian tube. Furthermore, PSGL-1 antigen gen was detected sporadically on microvascular endothelium in some pathologic tissues. This suggests that PSGL-1 may have functions other than mediating leukocyte adhersion.

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