The Impact of Rhg-CSF on the LFA-1 Conformation of CD4+T Cells in Mobilized Hematopoietic Stem Cell Allografts

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3464-3464
Author(s):  
Chunji Gao ◽  
Weihua Chen ◽  
Fei Wang ◽  
Meng Li ◽  
Haiyan Zhu ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Hematopoietic Stem ◽  
High Affinity ◽  
Before And After ◽  
Adhesion Activity ◽  
The Impact

Abstract In hemotopoietic stem cell transplantation (HSCT), the primary effects of G-CSF on cells of the hemotopoietic system include stimulation of proliferation and differentiation of HSCs, acceleration of neutrophil reconstitution after HSCT and mobilization of bone marrow HSCs into the peripheral blood. In recent years, several investigators have unraveled that G-CSF-mediated immune regulation including the switching T cell cytokine secretion profile to Th2 response, the altering adhesion activity of CD4+T cells to ICAM-1 and so on. Most of these cellular interactions of T cells are dependent on the integrin leukocyte function associated antigen-1 (LFA-1, αLβ2, CD11a/CD18) binding to intercellular adhesion molecule-1 (ICAM-1). Integrin αLβ2 adopts a bent, compact conformation in their low affinity state and an extended conformation in the high affinity state. The epitope of MEM148 is only expressed on the free β2 integrin subunit but is masked on LFA-1. The monoclonal antibody MEM148 reacts with an epitope exposed on free human CD18 chain as well as on high affinity state of LFA-1 that represents a reporter for αLβ2 active conformation. Our previous studies have demonstrated that rhG-CSF mobilization decreased the adhesion activity of CD4+T cells to ICAM-1, but did not find that rhG-CSF mobilization had effect on the level of CD11a expression. rhG-CSF maybe has effect on conformational change of LFA-1 on CD4+T cells. In order to explore the impact of rhG-CSF mobilization on LFA-1 conformation of CD4+ T cells, human CD4+T cells were isolated from peripheral blood mononuclear cells by positive selection with Miltenyi MACS. Isolated CD4+T cells were activated by OKT3+ICAM-1 and PMA+Ion separately. The slides with CD4+T cells were incubated with FITC-CD11a and FITC-MEM148 mAb, then were examined by using fluorescence microscope. The results show that the level of CD25, CD69 expression and MEM148 epitope exposure on activated CD4+T cells were significantly higher compared with unactivated CD4+T cells. rhG-CSF mobilization had no effect on the expression of CD11a, but could decrease expression of CD25, CD69 and exposure of MEM148 epitope on activated CD4+T cells. The percentage of LFA-1 polarized CD4+T cells before and after rhG-CSF mobilization was 85.32%, 61.86% respectively (p <0.01). No MEM148 epitope exposure on unactivated CD4+T cells was detected, but MEM148 epitope exposure on activated CD4+T cells before and after rhGCSF mobilization was 63.63%, 32.79% respectively (p <0.01). Overall, these data suggest that rhG-CSF can affect on the activation of CD4+T cells in mobilized hematopoietic stem cell allografts by altering the conformation of LFA-1.

scholarly journals Phosphorylated ERK1/2 in CD4 T cells is associated with acute GVHD in allogeneic hematopoietic stem cell transplantation

2020 ◽  
Vol 4 (4) ◽  
pp. 667-671
Author(s):  
Hidekazu Itamura ◽  
Takero Shindo ◽  
Satoshi Yoshioka ◽  
Takayuki Ishikawa ◽  
Shinya Kimura
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Acute Gvhd ◽  
Hematopoietic Stem

Abstract To diagnose graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is sometimes difficult. We showed previously that MEK inhibitors selectively suppress murine GVHD while retaining antiviral and antitumor immunity. Here, we asked whether the RAS/MEK/ERK pathway is activated in human allo-HSCT recipients with GVHD, and whether the phosphorylated ERK1/2 can be a biomarker of GVHD. Peripheral blood was sequentially collected from 20 allo-HSCT recipients: 1 bone marrow transplant, 7 peripheral blood stem cell transplants (PBSCT), and 12 cord blood transplants. Ten of the 20 allo-HSCT recipients developed GVHD, and phosphorylation of ERK1/2 in T and B cells was analyzed by flow cytometry. Occurrence of acute GVHD was associated with phosphorylation of ERK1/2 in CD4+ T cells at day 30 (P &lt; .001), which was suppressed by ex vivo exposure to a MEK inhibitor trametinib at clinically achievable concentrations. In particular, ERK1/2 was phosphorylated preferentially in naive/central memory CD4+ T cells. Notably, phosphorylation of ERK1/2 fell as GVHD improved. These results suggest that phosphorylation status of ERK1/2 in peripheral blood CD4+ T cells may be a future biomarker for diagnosing human GVHD, and the potential efficacy of MEK inhibitors against human GVHD.

Hematopoietic Stem Cell Differentiation Pathway in Humans Deduced From the Lineage Diversity of PNH-Type Cells in Patients with Bone Marrow Failure.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1489-1489
Author(s):  
Takamasa Katagiri ◽  
Zhirong Qi ◽  
Yu Kiyu ◽  
Naomi Sugimori ◽  
J. Luis Espinoza ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Bone Marrow Failure ◽  
Stem Cell Differentiation ◽  
Refractory Anemia ◽  
Hematopoietic Stem

Abstract Abstract 1489 Poster Board I-512 The hematopoietic stem cell (HSC) differentiation pathway in humans remains largely unknown due to the lack of an appropriate in vivo assay allowing the growth of HSCs as well as of clonal markers that enable the tracing of their progenies. Small populations of blood cells deficient in glycosylphosphatidylinositol-anchored proteins (GPI-APs) such as CD55 and CD59 are detectable in approximately 50% of patients with aplastic anemia (AA) and 15% of patients with refractory anemia (RA) of myelodysplastic syndrome defined by the FAB classification. Such blood cells with the paroxysmal nocturnal hemoglobinuria (PNH) phenotype (PNH-type cells) are derived from single PIGA mutant HSCs and their fate depends on the proliferation and self-maintenance properties of the individual HSCs that undergo PIG-A mutation by chance (Blood 2008;112:2160, Br J Haematol 2009 in press) Analyses of the PNH-type cells from a large number of patients on the diversity of lineage combination may help clarify the HSC differentiation pathway in humans because PIG-A mutant HSCs in patients with bone marrow failure appear to reflect the kinetics of healthy HSCs. Therefore, different lineages of peripheral blood cells were examined including glycophorin A+ erythrocytes (E), CD11b+ granulocytes (G), CD33+ monocytes (M), CD3+ T cells (T), CD19+ B cells (B), and NKp46+ NK cells (Nk) from 527 patients with AA or RA for the presence of CD55−CD59− cells in E and G, and CD55−CD59−CD48− cells in M,T, B, Nk with high sensitivity flow cytometry. Two hundred and twenty-eight patients (43%) displayed 0.003% to 99.1% PNH-type cells in at least one lineage of cells. The lineage combination patterns of PNH-type cells in these patients included EGM in 71 patients (31%), EGMTBNk in 43 (19%), EG in 37 (16%), T alone 14 (6%), EGMBNk in 11 (5%), G alone in 10 (4%), GM in 10 (4%), EGMNk in 7 (3%), EGMT in 7 (3%), EGMB in 6 (3%), EM in 5 (2%), EGMTB in 3 (1%), EGNk in 1 (0.4%), EGMTNk in 1 (0.4%), GMTB in 1 (0.4%), and GT in 1 (0.4%) (Table). All patterns included G or M, except for 14 patients displaying PNH-type T cells alone. No patients showed TB or TBNk patterns suggestive of the presence of common lymphoid progenitor cells. Peripheral blood specimens from 123 patients of the 228 patients possessing PNH-type cells were examined again after 3 to 10 months and all patients showed the same combination patterns as those revealed by the first examination. PIG-A gene analyses using sorted PNH-type cells from 3 patients revealed the same mutation in G and Nk for 1 patient and in G and T for 2 patients. These findings indicate that human HSCs may take a similar differentiation pathway to that of murine HSCs, the ‘myeloid-based model’ that was recently proposed by Kawamoto et al. (Nature 2008; 10:452), though the cases with PNH-type T cells alone remain to be elucidated. Table. Lineages of cells containing PNH-type cells in patients with AA or RA. The number in the parenthesis denotes the proportion of patients showing each combination pattern in the total patients possessing PNH-type cells. (+ ; presence of PNH-type cells) Disclosures No relevant conflicts of interest to declare.

Evaluation Of Immunocompentence In Tolerant Chimeric Recipients Of Hematopoietic Stem Cell/Renal Transplants

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4483-4483
Author(s):  
Joseph Leventhal ◽  
Larry D Bozulic ◽  
Mark D. Badder ◽  
Mary Jane Elliott ◽  
Michael N Issa ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell ◽  
Peripheral Blood ◽  
High Frequency ◽  
Blood Type ◽  
Phase 2 ◽  
Hematopoietic Stem ◽  
Tcr Repertoire

A phase 2 protocol was developed to attempt to induce donor-specific tolerance to renal allografts in related and unrelated donor/recipient combinations (IND 13947 phase 2). Subjects were conditioned with fludarabine (days -5, -4, -3), cyclophosphamide (50 mg/ky days -3 and +3), and 200 cGy total body irradiation. The kidney transplant was performed day 0. G-CSF mobilized peripheral blood stem cells processed to remove GVHD-producing cells and retain graft facilitating cells (FC) was administered on day +1. The conditioning was well tolerated and the subjects were managed as outpatients after post-operative day 2. This has resulted in high levels of durable chimerism and immunosuppression-free graft survival without GVHD or engraftment syndrome in mismatched related and unrelated recipients of living donor FC/hematopoietic stem cell/kidney allografts. Nine subjects are completely off immunosuppression from 2 months to 3 years. A number of others are in the process of tapering. In the present study, we have prospectively analyzed recovery of immune function, persistence of vaccination memory, and response to vaccination in subjects who exhibit high levels of chimerism. Chimerism testing was performed using molecular short tandem report (sensitivity ±5%). Three of four subjects who had been vaccinated to hepatitis B prior to transplantation and whose donors had not been vaccinated retained their immunity following transplantation. All subjects tested exhibited memory for varicella and the majority did as well for measles (9/11), mumps (8/11), and rubella (5/10). A blood group disparity was present in 9 chimeric donor/recipient pairs. One chimeric subject converted to donor blood type, 3 exhibited mixed donor/host RBC chimerism, and 5 retained their own blood type. Six chimeric subjects have been immunized with pneumococcal vaccine after transplantation. All generated an immune response to vaccination, confirming immunocompetence to generate an antibody response to antigen. Notably, recovery of CD8+ and CD4+ central memory, naïve, and effector memory T cells occurred within one year post-transplantation to levels that were not significantly different from pre-transplantation. In addition, CD31+/CD45RA+ CD8+ and CD4+ T cells representative of recent thymic emigrants were present by 3 months, demonstrating de novo thymic production of T cells after transplantation. Four patients were randomly selected for study of Tcell repertoire (TCR) generation after transplantation. Two had achieved durable full donor chimerism and the other two did not have durable chimerism. Peripheral blood samples freshly obtained from donors and recipients and T cell subsets were isolated using MACs microbead system. DNA was extracted and sequenced by ImmunoSeq (Adaptive Biotech, Seattle, WA) to evaluate TCR clonal repertoires in recipients. Although clonal diversity in TCR repertoire was reduced in post-Tx recipients (0.9 ± 0.05 pre-Tx vs. 0.79 ± 0.09 post-Tx), the repertoire was diverse enough to suggest recovery of immune competence. Interestingly, at least 97% of the unique sequences observed in post-Tx recipient were not present in either donor or recipient pre-Tx. Within the pool of shared sequences, full chimerism correlated with a shift towards homology with the donor, while loss of chimerism correlated with recipient pre-Tx. In addition, the chimeric patients also exhibited reduced diversity of TCR sequences and increased clonality. Top 20 “high frequency” clones are most stably expressed. CD8+ cells had the highest number of “high frequency” clones. Notably, the pattern of “low frequency” clones was highest in the CD127-CD4+CD25+ regulatory T cell subset, indicating an extensive and rapidly changing TCR repertoire. Taken together, these data suggest that immunologic recovery is robust in these nonmyeloablatively conditioned tolerant chimeric subjects. Disclosures: Bozulic: Regenerex, LLC: Employment. Badder:Regenerex, LLC: Employment. Ildstad:Regenerex, LLC: Equity Ownership.

scholarly journals Differential rates of replacement of human dermal dendritic cells and macrophages during hematopoietic stem cell transplantation

2009 ◽  
Vol 206 (2) ◽  
pp. 371-385 ◽  
Author(s):  
Muzlifah Haniffa ◽  
Florent Ginhoux ◽  
Xiao-Nong Wang ◽  
Venetia Bigley ◽  
Michal Abel ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Cd4 T Cells ◽  
Critical Role ◽  
Hematopoietic Stem ◽  
Memory Cd4 T Cells

Animal models of hematopoietic stem cell transplantation have been used to analyze the turnover of bone marrow–derived cells and to demonstrate the critical role of recipient antigen-presenting cells (APC) in graft versus host disease (GVHD). In humans, the phenotype and lineage relationships of myeloid-derived tissue APC remain incompletely understood. It has also been proposed that the risk of acute GVHD, which extends over many months, is related to the protracted survival of certain recipient APC. Human dermis contains three principal subsets of CD45+HLA-DR+ cells: CD1a+CD14− DC, CD1a−CD14+ DC, and CD1a−CD14+FXIIIa+ macrophages. In vitro, each subset has characteristic properties. After transplantation, both CD1a+ and CD14+ DC are rapidly depleted and replaced by donor cells, but recipient macrophages can be found in GVHD lesions and may persist for many months. Macrophages isolated from normal dermis secrete proinflammatory cytokines. Although they stimulate little proliferation of naive or memory CD4+ T cells, macrophages induce cytokine expression in memory CD4+ T cells and activation and proliferation of CD8+ T cells. These observations suggest that dermal macrophages and DC are from distinct lineages and that persistent recipient macrophages, although unlikely to initiate alloreactivity, may contribute to GVHD by sustaining the responses of previously activated T cells.

scholarly journals IMMU-24. THE IMPACT OF BRAIN TUMORS ON HEMATOPOIETIC STEM CELL-DERIVED T CELLS

2018 ◽  
Vol 20 (suppl_6) ◽  
pp. vi126-vi126
Author(s):  
Bayli DiVita Dean ◽  
Brandon Fernandez ◽  
Delaney Woodworth ◽  
Tyler Wildes ◽  
Duane Mitchell ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Brain Tumors ◽  
Hematopoietic Stem Cell ◽  
Hematopoietic Stem ◽  
The Impact

Thymic Dendritc Cells Generated Ex Vivo Induce Donor Regulatory T Cell Differentiation and Hasten Immune Reconstitution After Hematopoietic Stem Cell Transplantation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1905-1905
Author(s):  
Mari Hashitate Dallas ◽  
Deanna Langfitt ◽  
Kenneth Busby
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Control Group ◽  
Hematopoietic Stem

Abstract Abstract 1905 Dendritic cells (DCs) are the first committed cells to engraft in the thymus after hematopoietic stem cell transplantation (HSCT). The critical role of thymic DCs in ensuring efficient tolerance and selection has been well demonstrated, however its role in facilitating donor engraftment has not been reported. Here we show DCs accelerates thymic reconstitution by inducing regulatory T cells (Tregs) differentiation and enhancing T cell recovery after HSCT. Lethally irradiated CD45.2 C57BL/6 control group received 103 CD45.1 lin−sca-1+c-kit+ (LSK) hematopoietic stem cell progenitors while the DCs group received 103 CD45.1 LSK cells along with 103 CD45.2 GFP+ DCs. DCs were generated ex vivo using bone marrow from CD45.2 GFP+ CD57BL/6 mice and cultured for 7 days with GMCSF. At 4 and 7 days after HSCT, the thymus of DC group contained 1.8 and 4.2- fold higher number of thymocytes (p<0.05) and a 3.2 and 7.4-fold, respectively, higher number of donor derived thymoctyes compared to the control group (p<0.05). Moreover, thymuses of the DCs group had GFP+ CD11c+ cells present in the medulla and 5.6-fold increase in the number of donor derived FoxP3+ Tregs compared to control confirmed by immunohistochemistry (IHC). Furthermore, thymic recovery scored by a pathologist blinded to the groups found significant increase in lymphoid regeneration (H&E) and higher number of CD3+ lymphoid aggregates (IHC) in the DC group compared to control group that had severe, diffuse lymphoid depletion. Lastly, at 2 and 4 weeks after HSCT, peripheral blood of DCs group contained 2.6 and 4.8-fold, respectively, higher numbers of CD3+ cells derived from donor LSK cells compared to the control group (p<0.05). Here, we demonstrate that donor DCs efficiently migrate and home to the thymic medulla and hasten thymic recovery as demonstrated by the higher number of total thymoctyes. Furthermore, DCs facilitate thymic engraftment as shown by increase number of donor derived FoxP3+ Tregs and thymocytes. Lastly, recipients of DCs have earlier generation of de-novo donor derived CD3+ T cells in the peripheral blood. By using the GFP+ cells along with donor LSK cells, we were able to confirm that the facilitation of early thymic recovery was due to the increased engraftment of the donor cells rather than autologous recovery of the host. In conclusion, this study demonstrates that DCs committed prior to thymic entry maintains the ability to home to the medullary region and facilitate thymic recovery by enhancing Tregs differentiation. Thus, ex vivo generation of donor DCs to augment a HSC graft may Disclosures: No relevant conflicts of interest to declare.

Erythroid-Lineage Specific Engraftment in Patients with Severe Hemoglobinopathy Following Allogeneic Hematopoietic Stem Cell Transplantation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2996-2996
Author(s):  
Paul M. Armistead ◽  
Mehrdad Mohseni ◽  
Roslyn Gerwin ◽  
Masood Iravani ◽  
Bahram Chardouli ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Peripheral Blood ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
The Impact

Abstract The monitoring of lineage-specific engraftment is critical to understanding the impact of novel transplant regimens and determining how these can be modified to improve outcomes. We developed an RNA-based pyrosequencing assay to rapidly measure lineage-specific chimerism by quantification of cell type specific host versus donor transcripts that differ in the expression of single nucleotide polymorphisms (SNPs). To assess RBC lineage engraftment, we identified 10 common SNPs, expressed by 7 genes that encode RBC specific antigens and structural proteins by using the HapMap and Ensembl databases and direct high-throughput genotyping. These SNPs could then be PCR-amplified from total RNA extracted from peripheral blood, which contains nucleated erythroid progenitors. Mixing studies using samples of peripheral blood with defined alleles were performed to validate that each SNP could quantitatively measure donor/recipient DNA and RNA. Using this panel, we directly genotyped 15 patients and their HLA-matched related donors who underwent allogeneic hematopoietic stem cell transplantation for sickle cell disease (SCD) or thalassemia major. A median of 3 SNPs was informative for each donor/recipient pair. By using informative expressed RBC SNPs to quantify donor-derived RBC transcripts, we measured serial rates of erythroid lineage specific engraftment in 13 of the 15 patients that were compared to overall levels of donor mononuclear cell (WBC) engraftment. In pairs with greater than 1 informative SNP, high concordance in serial post-transplant chimerism measurements among individual SNPs was observed. At post-transplant day 30, 4 of 13 patients converted to full donor hematopoiesis, 1 demonstrated primary graft failure, and 8 developed partial donor WBC engraftment, ranging from 29 – 82%. Consistent with known ineffective erythropoiesis associated with SCD and thalassemia, we detected up to 3-fold greater RBC specific compared to overall WBC engraftment in 5 of 8 patients. In contrast, the remaining 3 of 8, all of whom received ABO-incompatible grafts, demonstrated at least 0.5-fold lower RBC compared to WBC engraftment. Detection of the effects of ABO incompatibility by RNA pyrosequencing was related to persistence of anti-isohemaglutinin antibodies. Since erythroid progenitors, the cell population evaluated by our assay, transit rapidly in peripheral blood relative to long-lived mature erythrocytes, RBC engraftment is potentially a sensitive marker for graft rejection. In keeping with this, 3 of 8 patients eventually rejected their grafts at 60, 219, and 288 days post-transplant, in which loss of WBC and RBC engraftment was concurrently detected. In summary, RNA pyrosequencing provides rapid measurement of erythroid lineage chimerism, without requiring specific cell isolation, and can provide valuable functional information for diseases in which RBC engraftment is critically important. Similar methods can be applied to generate panels of expressed SNPs informative for other cell lineages to assess the impact of novel stem cell therapies on lineage-specific engraftment.

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