scholarly journals Granulocyte Colony-Stimulating Factor Effectively Mobilizes TCR γδ and NK Cells Providing an Allograft Potentially Enhanced for the Graft-Versus-Leukemia Effect for Allogeneic Stem Cell Transplantation

2021 ◽  
Vol 12 ◽  
Author(s):  
Lia Minculescu ◽  
Henrik Sengelov ◽  
Hanne Vibeke Marquart ◽  
Lars Peter Ryder ◽  
Anne Fischer-Nielsen ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Cell Compartment ◽  
Effector Cells ◽  
Graft Versus Leukemia ◽  
Hla Dr

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potential cure for patients with hematological malignancies but substantial risks of recurrence of the malignant disease remain. TCR γδ and NK cells are perceived as potent innate effector cells in HSCT and have been associated with post-transplant protection from relapse in clinical studies. Immunocompetent cells from the donor are crucial for patient outcomes and peripheral blood stem cells (PBSC) are being increasingly applied as graft source. G-CSF is the preferential mobilizing agent in healthy donors for PBSC grafts, yet effects of G-CSF on TCR γδ and NK cells are scarcely uncovered and could influence the graft composition and potency of these cells. Therefore, we analyzed T and NK cell subsets and activation markers in peripheral blood samples of 49 donors before and after G-CSF mobilization and—for a subset of donors—also in the corresponding graft samples using multicolor flowcytometry with staining for CD3, CD4, CD8, TCRαβ, TCRγδ, Vδ1, Vδ2, HLA-DR, CD45RA, CD197, CD45RO, HLA-DR, CD16, CD56, and CD314. We found that TCR γδ cells were mobilized and harvested with an efficiency corresponding that of TCR αβ cells. For TCR γδ as well as for TCR αβ cells, G-CSF preferentially mobilized naïve and terminally differentiated effector (TEMRA) cells over memory cells. In the TCR γδ cell compartment, G-CSF preferentially mobilized cells of the nonVδ2 types and increased the fraction of HLA-DR positive TCR γδ cells. For NK cells, mobilization by G-CSF was increased compared to that of T cells, yet NK cells appeared to be less efficiently harvested than T cells. In the NK cell compartment, G-CSF-stimulation preserved the proportion of CD56dim NK effector cells which have been associated with relapse protection. The expression of the activating receptor NKG2D implied in anti-leukemic responses, was significantly increased in both CD56dim and CD56bright NK cells after G-CSF stimulation. These results indicate differentiated mobilization and altering properties of G-CSF which could improve the effects of donor TCR γδ and NK cells in the processes of graft-versus-leukemia for relapse prevention after HSCT.

The Studies on the T-Cell Receptor Repertoire Diversity and Flow Cytometric Analysis in a Patient with MAS/HPS after Unrelated Peripheral Blood Stem Cell Transplantation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4970-4970
Author(s):  
Xin Du ◽  
Yangqiu Li ◽  
Suxia Geng ◽  
Jianyu Weng ◽  
Zesheng Lu ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Peripheral Blood Stem Cell ◽  
Nk Cell ◽  
Flow Cytometric Analysis ◽  
Blood Stem Cell ◽  
Flow Cytometric

Abstract Macrophage activation syndrome (MAS) /Hemophagocytic syndrome (HPS) is characterized by proliferation of activated macrophages under conditions such as infection(C Clin Infect Dis 2004)lymphoma(Aouba A Am J Hematol 2004), autoimmune disease(Kaneko K Clin Rheumatol 2005), solid organ transplantation(Akamatsu N,Transplant Proc 2006;). There have been several reports of MAS /HPS after hematopoietic stem cell transplantation, involving not only allogeneic,but also autologous transplantation(Sreedharan A Bone Marrow Transplantation,2006). Generally, MAS /HPS is a cytokine-related disorder.But at present, its clinical characteristics remain unknown. We firstly study here the T-cell receptor repertoire diversity and flow cytometric analysis in MAS /HPS after unrelated peripheral blood stem cell transplantation. The CDR3 of TCR Vα and Vβ subfamily genes were amplified in peripheral blood mononuclear cells from the patient with MAS/HPS after unrelated peripheral blood stem cell using RT-PCR for detection of the distribution of TCR Vα and Vβ repertoire, the PCR products were further analyzed by genescan technique for the CDR3 size, to evaluating clonality of the detectable TCR Vα and VβT cells. Lymphocyte subsets in the peripheral blood were detected by monoclone antibody and flow cytometry including T lymphocyte subsets and NK cells. Flow-cytometric analysis showed CD56+ CD16+ cell 68.65% and CD3+ cell 11.79% in the lymphocyte population;CD16+CD69+ cell 68.51% and CD25+CD16+ cell 31.59% in NK cell. In the T lymphocytic subsets, CD25 + CD3+ cell 62%; CD69+CD3+ cell 75.81%; CD25CD4+ cell 0.81%,CD25CD8+ cell 3.48%; CD69CD4+ cell 0.31%, CD69+CD8+ cell 16.86%.The results show that the main activated lymphocytes is NK cell in patient at diagnosed with MAS/HPS. Of interest, it was only after the addition of high-dose IVIG 1g/kg/d for two days (Ostronoff et al BMT2006) to the treatment that MAS remitted. There are 23 Vα and 15Vβ subfamily T cells could be identified in this time, and the clonal expansion T cells could be found in TCR Vα5, 13, 20; TCR Vβ4, 11, 15 and 21subfamilies. Billiau et al (Blood 2005)describes the immunohistochemical findings on liver tissues from 5 children with MAS in the context of a different type of hemophagocytic syndrome (HPS) in liver transplantation. This study is the first directly to substantiates the presumed immunopathogenesis of MAS by documenting in situ expression of IFN-γ+ by activated CD8+ lymphocytes, and of IL-6 and TNF-α+ by hemophagocytosing macrophages, on liver tissues of patients with MAS. We found no evidence of potential infectious, autoimmune or malignant triggers of R-HPS in our patient, despite extensive investigations. We conclued that the skew distribution and clonal expansion of TCR Vα and Vβ subfamily T cells underscore the primary role of T cells in the pathogenesis of MAS/HPS.

Myeloid Chimerism Reflects Engraftment of Donor Hematopoiesis, Whereas T Cell Chimerism Reflects Survival and Expansion of Donor and Recipient Residual Mature T Cells Early After T Cell Depleted Allogeneic Stem Cell Transplantation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4475-4475
Author(s):  
Jessica C. Harskamp ◽  
Esther H.M. van Egmond ◽  
Hans L. Vos ◽  
Stijn J.M. Halkes ◽  
Roel Willemze ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Median Frequency ◽  
Cell Compartment ◽  
Hematopoietic Lineage ◽  
Chimerism Analysis

Abstract Abstract 4475 Allogeneic stem cell transplantation (alloSCT) is frequently complicated by life-threatening graft versus host disease (GVHD). Previous studies demonstrated that T cell depletion (TCD) of the graft significantly decreases the incidence and severity of GVHD, and is associated with a higher percentage of patients with mixed chimerism (MC). In most studies chimerism analysis is performed on the total bone marrow (BM) leukocyte fraction, and changes in chimerism are related to engraftment. In this study we investigated whether MC in the total BM leukocyte fraction truly reflects engraftment or if it is influenced by survival and expansion of donor and recipient residual mature T cells, and whether hematopoietic lineage specific chimerism analysis is therefore a better method to determine engraftment. It is likely that chimerism analysis of the stem cell compartment is best reflected in peripheral blood (PB) in those cells that are continuously produced and short lived, such as monocytes and granulocytes, and therefore PB myeloid chimerism primarily reflects engraftment. In contrast, previous studies have shown by T cell receptor excision circle analysis that T cell neogenesis is virtually absent in the first 6 months after alloSCT, and that predominantly memory T cells are present in PB and BM. Therefore, we hypothesize that MC of these long lived T cells merely reflects survival and expansion of recipient and donor residual T cells. Since the life span of B and NK cells is longer than myeloid cells, but shorter than T cells, we anticipate that in the first 6 months after alloSCT, B and NK cell chimerism reflects a combination of survival and neogenesis. To analyze these hypotheses we performed hematopoietic lineage specific chimerism analysis on PB cells of 22 patients (median age 52 years, range 23-73, 11 males) receiving a TCD alloSCT between June and November 2008 after a myeloablative (n=11) or non myeloablative conditioning regimen (n=11) for AML, ALL, high risk MDS, multiple myeloma, CML, CLL or NHL. At intervals of 6 weeks PB was collected, and monocytes, granulocytes, B and NK cells, CD4+ and CD8+ T cells were sorted. The total leukocyte fraction was obtained by erythrocyte lysis of BM. DNA was isolated to perform chimerism analysis using short tandem repeats - PCR. Our results show that in the BM leukocyte fraction 47% of the patients were MC at 3 months after alloSCT, with a median frequency of patient cells of 4%. However, of the patients with MC in the total leukocyte fraction, 67% was complete chimeric in the myeloid subsets and MC in the T cell compartment. In the PB myeloid subsets (monocytes and granulocytes) less than 28% of the patients were MC during the first 6 months after alloSCT with a median frequency of patient cells less than 5%. In the B and NK cell subsets, at most time points more patients were MC (7-43%) with higher frequencies of patient cells (2-14%) compared to the myeloid subsets. The CD4 and CD8 T cell subsets showed the highest frequencies of MC in numbers of patients (31-61%) as well as the highest MC frequencies of patient cells (13-80%). Phenotypic analysis of the T cell compartment showed that 98% of the CD4 and CD8 T cells were memory cells during the first 6 months after alloSCT. Preliminary data indicate that the median percentage of donor derived T cells increased during the first 6 months after alloSCT, correlating with development of mild GVHD, suggesting that T cell chimerism is influenced by immunogenic triggers. In conclusion, these results illustrate that for engraftment and neogenesis of donor hematopoiesis, myeloid chimerism analysis provides more accurate information than total BM leukocyte chimerism analysis, since the results are greatly influenced by T cell chimerism. Since almost all T cells were memory cells within the first 6 months after alloSCT, T cell chimerism analysis reflects survival and expansion of mature donor as well as recipient T cells, and can therefore not be used to measure engraftment. Disclosures: No relevant conflicts of interest to declare.

CD16+CD56− NK Cells in the Peripheral Blood of Cord Blood Transplant Recipients: A Unique Subset of Immature NK Cells Possibly Associated with Graft-Versus-Leukemia Effect.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1074-1074 ◽  
Author(s):  
Xuzhang Lu ◽  
Yokio Kondo ◽  
Hiroyuki Takamatsu ◽  
Hiroshita Yamazaki ◽  
Zhirong Qi ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cord Blood ◽  
Nk Cells ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Flow Cytometric Analysis ◽  
Cell Subset ◽  
Leukemic Cells

Abstract NK cells play a major role in the activity of graft-versus-host (GVL) effect after an HLA-mismatched stem cell transplantation. In unrelated cord blood transplantation (CBT) where there is often an HLA mismatch between the donor and recipient, NK cells may also play a vital role, though their roles have not been extensively studied. Cord blood (CB) is known to have a unique subset of NK cells characterized by a CD16+CD56− phenotype. CD16+CD56− NK cells in CB are thought to be progenitors of CD16+CD56+ NK cells because CD16+CD56− NK cells acquires CD56 expression after in vitro culture in the presence of IL-2. However, the function of this immature NK cell subset after CBT remains unknown. A marked increase in the number of CD16+CD56- NK cells in the peripheral blood of an HLA-mismatched CBT recipient with acute myeloid leukemia (AML) was recently observed. A 56-year old male, who received a reduced intensity CBT following a full relapse after allogeneic stem cell transplantation from an HLA-matched sibling donor, showed an increase in the copy number of WT-1 mRNA in the peripheral blood around day 80 after the CBT, but the WT-1 copy number decreased from 1500/microliter RNA to 230/microliter RNA in association with the increase in the number of CD16+CD56- NK cells, and his molecular remission lasted more than 1.5 years thereafter. This case prompted an investigation of CD16+CD56− NK cells in the peripheral blood after allogeneic stem cell transplantation. A similar increase in the proportion of CD16+CD56− NK cells (20% or more) in the peripheral blood CD16+ NK cells was observed in 64% (7/11) of CBT recipients, all of whom maintained remission, but in none of the 11 bone marrow and 8 peripheral blood stem cell transplant recipients examined (Figure 1). CD16+CD56− NK cells in CBT recipients expressed receptors specific to NK cells such as NKp30 and NKp46 same level as CD16+CD56− NK cells of fresh CB cells. CD16+CD56− NK cells isolated from CBT recipients became CD56+ when they were cultured in the presence of IL-2 with or without K562-mb15-4-1BBL. When cultured NK cells derived from the CD16+CD56− NK cells were separated into CD158b+ and CD158b− cells, CD158b+ cells failed to kill 721–221 cells transfected with HLA-C*0301 while they killed untransfected or HLA-C*0401-transfected 221 cells. Despite the presence of the corresponding KIR ligand (C*0304), cultured CD16+CD56− NK cells showed cytotoxicity against the patient’s leukemic cells. These findings suggest that an increase in the proportion of CD16+CD56− NK cells is unique to recipients of CBT and that this immature NK-cell subset in CBT recipients may undergo differentiation into mature NK cells in vivo capable of killing residual leukemic cells, thereby contributing to the GVL effect regardless of the presence of the KIR ligand. Figure 1 Flow cytometric analysis of CD3-CD16+CD56-cells in peripheral blood of SCT recipients and healthy individual.Examples of three-flourescence cytofluorometric analysis of fresh isolated PBMC stained with CD3,CD56 and CD16 in different SCT patients and health individuals. The characterization of the unusual CD56-CD16+ cell subset expend only in the CBT individual(a). Presenting cellware gated on CD3-cells Figure 1. Flow cytometric analysis of CD3-CD16+CD56-cells in peripheral blood of SCT recipients and healthy individual.Examples of three-flourescence cytofluorometric analysis of fresh isolated PBMC stained with CD3,CD56 and CD16 in different SCT patients and health individuals. The characterization of the unusual CD56-CD16+ cell subset expend only in the CBT individual(a). Presenting cellware gated on CD3-cells

scholarly journals NK Cell Reconstitution in Paediatric Leukemic Patients after T-Cell-Depleted HLA-Haploidentical Haematopoietic Stem Cell Transplantation Followed by the Reinfusion of iCasp9-Modified Donor T Cells

2019 ◽  
Vol 8 (11) ◽  
pp. 1904 ◽  
Author(s):  
Helena Stabile ◽  
Paolo Nisti ◽  
Cinzia Fionda ◽  
Daria Pagliara ◽  
Stefania Gaspari ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplantation ◽  
Nk Cells ◽  
Cell Transplantation ◽  
Nk Cell ◽  
Haematopoietic Stem Cell ◽  
Hematopoietic Stem ◽  
Donor T Cells

T-cell-depleted (TCD) human leukocyte antigen (HLA) haploidentical (haplo) hematopoietic stem cell transplantation (HSCT) (TCD-haplo-HSCT) has had a huge impact on the treatment of many haematological diseases. The adoptive transfer of a titrated number of T cells genetically modified with a gene suicide can improve immune reconstitution and represents an interesting strategy to enhance the success of haplo-HSCT. Natural killer (NK) cells are the first donor-derived lymphocyte population to reconstitute following transplantation, and play a pivotal role in mediating graft-versus-leukaemia (GvL). We recently described a CD56lowCD16low NK cell subset that mediates both cytotoxic activity and cytokine production. Given the multifunctional properties of this subset, we studied its functional recovery in a cohort of children given α/βT-cell-depleted haplo-HSCT followed by the infusion of a titrated number of iCasp-9-modified T cells (iCasp-9 HSCT). The data obtained indicate that multifunctional CD56lowCD16low NK cell frequency is similar to that of healthy donors (HD) at all time points analysed, showing enrichment in the bone marrow (BM). Interestingly, with regard to functional acquisition, we identified two groups of patients, namely those whose NK cells did (responder) or did not (non responder) degranulate or produce cytokines. Moreover, in patients analysed for both functions, we observed that the acquisition of degranulation capacity was not associated with the ability to produce interferon-gamma (IFN-γ Intriguingly, we found a higher BM and peripheral blood (PB) frequency of iCas9 donor T cells only in patients characterized by the ability of CD56lowCD16low NK cells to degranulate. Collectively, these findings suggest that donor iCasp9-T lymphocytes do not have a significant influence on NK cell reconstitution, even if they may positively affect the acquisition of target-induced degranulation of CD56lowCD16low NK cells in the T-cell-depleted haplo-HSC transplanted patients.

scholarly journals Characterization of Peripheral Blood Mononuclear Cells Addback Following CD34 Enrichment, Engraftment and T and NK Cells Immune Reconstitution in Patients with High Risk Sickle Cell Disease (SCD) (IND 14359)

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 20-21
Author(s):  
Yaya Chu ◽  
Julie-An Talano ◽  
Lee Ann Baxter-Lowe ◽  
Carolyn A. Keever-Taylor ◽  
Erin Morris ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Immune Reconstitution ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Advisory Committees ◽  
Hla Dr

Background: CD3/CD19 cell depletion (Barfiled RC, et al, Cytotherapy, 2004), αβ T-cell/CD19 cell depletion (Locatelli F, et al, Blood, 2017), CD34+ positive selection (Aversa F, et al, NEJM, 1998) are designed to deplete T cells and reduce AGVHD following allogeneic stem cell transplantation (AlloSCT). These approaches achieved low rates of AGVHD, but the grafts had few T and B cells. To improve immune reconstitution we undertook an alternative approach to addback small numbers and percentages of immune cells in the final HSCT product. We previously reported a very low incidence of AGVHD in pediatric recipients receiving CD34 enriched HPC products with peripheral blood mononuclear cells (PBMNC) addback containing a fixed dose of 2 x 105 CD3/kg from MUD donors (Geyer/Cairo et al, BJH, 2012). Recently we demonstrated that despite a 5 log depletion of T cells, PBMNC addback (fixed at 2 x 105 CD3/kg) facilitated rapid hematopoietic engraftment, high levels of donor chimerism and immune reconstitution with a low probability of Grade II-IV AGVHD. Patients had a 1 yr OS of 90% following familial haploidentical (FHI) CD34 Enriched Stem Cell Transplantation in patients with SCD (Cairo, JAMA Pediatr, 2020). Objective: To determine the final immune cell concentration following CD34 enrichment and PBMNC (2 x 105 CD3/kg) addback and determine the effect on engraftment and T and NK cell immune reconstitution. Methods: Patients and/or their guardians signed written informed consents and/or assents (NCT NCT02675959). CD34+ enrichment was performed using a CD34+ reagent system (CliniMACS; Miltenyi Biotec). Mononuclear cells (2 × 105 CD3 cells/kg of recipient body weight) were removed from the leukapheresis collection prior to CD34+ enrichment and were cryopreserved as a source of MNC addback (T cells). The addback products were analyzed for CD3+CD56- T cells, CD3-CD56+ NK cells, CD3+CD56+ NKT cells, Lin-CD123+ HLA-DR+ DC cells and Lin-CD11c+ HLA-DR+ DC cells by multicolor flow cytometry analysis. Th1/Th2 cytokines were measured by multiplex assays. T cell activity was measured by viral T cells IFN-g and plasma cytokines. NK function was measured by NK receptor expression by flow cytometry analysis and in vitro cytotoxicity. Results: We identified in the PBMNC addback, mean+SEM white blood cell (WBC) percentage of: CD3+ CD56- T cells = 56.4±5%; CD3- CD56+ NK cells = 4.6±1%; CD3+ CD56+ NKT cells = 5.1±0.6%; CD19+ B cells = 29.9±3.5%. Lin- WBC consisted of: CD123+ HLA-DR+ DC cells = 18.4±8.2%; CD11c+ HLA-DR+ DC cells = 6.0±3.0%. There were 20.0+9.1e6 T cells, 1.1+0.3e6 NK cells, 1.6+0.7 e6 NKT cells, 8.6+2.5e6 B cells, 1.2+0.6e6 CD123+DC and 0.8+0.5e6 CD11c DC in the final infused products (Fig.1). We found that percentages of IFN-g+ in CD4 cells in response to CMV (pp65), ADV (hexon) and EBV (BZLF1), ranged from 0.2%+0.1% to 0.5%+0.1%, while percentages of IFN-g+ in CD8 cells in response to the antigens ranged from 0.7%+0.3% to 3.7%+1.8% when examined at days 180, 270 and 365. NK (CD3- CD56+) reconstitution was extremely rapid and occurred as early as day 30 (35.5±8.6%, 2710+1624.4 cells/ul total cells; p<0.01 vs pre-t). There were no significant differences pre-HSCT vs day 365 in plasma cytokines (Th1 and Th2) and growth factors released including IFN-g, TNF-a, IL-18, IL-4, IL-5, IL-6, IL-10, G-CSF, MCP-1 and MIP1a. There was also robust expression of NK receptor expression including NK cytotoxicity receptors, NK KIR receptors, and C-type lectin-like receptors at day 30 as compared to pre-HSCT. NK cytotoxicity, as measured using PBMC cells from recipients at different time points against K562 (E:T=10:1), was also significantly increased at day 30 (26.2±2.8%) and day 180 (28.3±3%) vs pre-HSCT (16.1±2.1%) (p<0.01). As a NK cell activation marker, CD107a expression and granzyme B levels in gated NK cells peaked at day 30. Conclusion: PBMNC addback to CD34 enriched HPC products, with a final dose of 2 × 105 CD3 cells/kg, led to stem cell products with a diverse mixture of T, NK, NKT, DC1, and DC2 cells. Immune reconstitution following PBMNC addback to CD34 enriched cells resulted in excellent CD4 and CD8 responses to CMV, ADV and EBV, and rapid functional NK cell reconstitution (Supported by FDA R01FD004090 (MSC)). Disclosures Baxter-Lowe: CHLA: Current Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Patents related to HLA typing, Research Funding. Johnson:Miltenyi Biotec: Research Funding; Cell Vault: Research Funding. Cairo:Miltenyi: Research Funding; Technology Inc/Miltenyi Biotec: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nektar Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Repopulation of Lymphocytes and Natural Killer Cells on Day 15 Following First Autologous Stem Cell Transplantation in Myeloma Patients Correlates with the Number of Reinfused Lymphocytes and Natural Killer T Cells in the Autologous Graft

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5820-5820
Author(s):  
Matevz Skerget ◽  
Barbara Skopec ◽  
Samo Zver
Keyword(s):  
Body Weight ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Induction Treatment ◽  
Cd34 Cells ◽  
Lymphocyte Number

Abstract Introduction Autologous hematopoietic stem cell transplantation (aHSCT) is still considered standard of care for eligible myeloma patients following induction treatment. Beside the effect of a high dose melphalan, several studies proposed a beneficial effect of immune graft versus myeloma effect following aHSCT. The higher number of lymphocytes and natural killer (NK) cells on day 15 after aHSCT correlated with a better longterm outcome. We have previously published the data on the influence of different mobilization regimens on the number of collected lymphocytes and NK cells and their number on day 15 post aHSCT. In this abstract we present the data on the correlation between the dose of reinfused lymphocytes, NK cells and CD34+ cells and the number of lymphocytes and NK cells on day 15 after aHSCT. Methods We prospectively enrolled 48 newly diagnosed myeloma patients following induction treatment with bortezomib and dexamethasone. They were mobilized with either filgrastim (10 mcg/kg body weight daily), pegfilgrastim (12 mg one-time injection) or cyclophosphamide (4 g/m2) followed by filgrastim (10 mcg/kg body weight daily). The lymphocyte and NK cell (CD16+/56+) counts in the stem cell concentrate and on day 15 post aHSCT in peripheral blood were determined with the flow cytometer. Pearson r correlation was used to associate the dose of infused cells and their number on day 15 after aHSCT in peripheral blood. Scatter plots and linear regression were used for the data presentation. SPSS Statistics v21 (IBM, USA) was used for the analysis. The study complied with the Declaration of Helsinki and was approved by the local ethical committee. All patients signed a written informed consent. Results We established a correlation between the dose of infused lymphocytes in the autologous graft and their number in the peripheral blood on day 15 after aHSCT (Figure 1; Pearson r = 0.27, p = 0.04). There was also a correlation between the dose of infused NK cells and their number in the peripheral blood on day 15 post aHSCT (Figure 2; Pearson r = 0.6, p < 0.01). However, there was no correlation between the number of reinfused CD34+ cells and the number of lymphocytes on day 15 after aHSCT (Figure 3). Conclusion The early immune repopulation is important for better overall survival after the aHSCT in multiple myeloma and lymphoma patients. The data from our study showed that the higher numbers of reinfused lymphocytes and NK cells, but not the total number of CD 34+ cells, significantly impact earlier lymphocyte and NK cell engraftment. We concluded that with the aim to achieve the best possible outcomes of aHSCT for our patients, we must make sure to reinfuse not only sufficient number of CD 34+ cells, but also of lymphocytes and NK cells. Figure 1 Correlation between a dose of the infused lymphocytes and their number in the peripheral blood on day 15 after aHSCT (Pearson r = 0.27, p = 0.04). Figure 1. Correlation between a dose of the infused lymphocytes and their number in the peripheral blood on day 15 after aHSCT (Pearson r = 0.27, p = 0.04). Figure 2 Correlation between the dose of the infused NK cells and their number in the peripheral blood on day 15 post aHSCT (Pearson r = 0.6, p < 0.01). Figure 2. Correlation between the dose of the infused NK cells and their number in the peripheral blood on day 15 post aHSCT (Pearson r = 0.6, p < 0.01). Figure 3 Correlation between the dose of infused CD34+ cells and the total lymphocyte number in the peripheral blood on day 15 post aHSCT. Figure 3. Correlation between the dose of infused CD34+ cells and the total lymphocyte number in the peripheral blood on day 15 post aHSCT. Disclosures Skopec: amgen: Consultancy, Honoraria; novartis: Honoraria.

High Numbers of NK Cell Clones Can Be Generated by Limiting Dilution in SCGM Medium Supplemented with IL-2, IL-15 and Anti-NKp46 Antibody 9E2.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3876-3876
Author(s):  
Sabine Kowald ◽  
Andreas Arendt ◽  
Jurgen Schmitz ◽  
Volker Huppert
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Nk Cells ◽  
Cell Transplantation ◽  
Cell Activation ◽  
Nk Cell ◽  
Cloning Efficiency ◽  
Cell Clones ◽  
Limiting Dilution

Abstract NK cell clones obtained by limiting dilution have frequently been used for functional studies and as an analytical tool e.g. for assessment of alloreactivity in the context of haploidentical stem cell transplantation [Velardi et al., Blood 2007]. NK cell clones can be used in co-culture experiments with allogeneic target cells to determine the number of alloreactive clones and therefore to quantify NK cell alloreactivity. This method provides additional quantitative data, while PCR based methods for HLA and KIR typing only provide qualitative results of potential alloreactivity. Generation of NK cell clones usually is difficult and cloning efficiency is low (&lt; 5% [Yssel et al., J. Immunol. Methods 1984]). Protocols have been developed to increase cloning efficiency [Wang et al., J. Immunol. Methods 2005] We evaluated whether cloning efficiency can be further increased by optimization of cell processing prior to culture and/or of medium components. Published reference methods use media supplemented with Interleukin-2 (IL-2). These conditions also stimulate proliferation of T cells, which usually overgrow NK cells in culture. Thus depletion of T cells prior to incubation with IL-2 may facilitate identifation of proliferating NK cells. T cells can be actively depleted upon CD3 magnetic labelling, by use of an NK cell isolation kit or passively by CD56 enrichment. CD 56 enrichment efficiently depletes CD3+CD56- T cells and residual CD3+CD56+ NKT cells do not expand under the chosen culture conditions. IL-15 is capable of preventing NK cell apoptosis at low concentrations [Caligiuri et al., J. Clin. Invest. 1997; Carson et al., J Clin Invest. 1997; Puzanov et al., J Immunol. 1996)] and plays a role in proliferation and survival of NK cells. Cross linking of NKp46 leads to NK cell activation [Long et al., Blood 2005]. As simultaneous engagement of two independent activating NK cell receptors (IL-2/IL-15 receptors and NKp46) may enhance NK cell activation and proliferation, we evaluated different combinations of IL-2, IL-15 and the anti-NKp46 antibody 9E2. Optimal results were obtained when CD56 selected cells were used (compared to CD3 depletion, NK cell enrichment, KIR2D selection). Supplementation of SCGM medium (Cell Genix) with IL-2 (500u/mL), IL-15 (10ng/mL) and anti NKp46 antibody (2μg/mL, CD335, clone 9E2) increased the number of NK cell clones by a factor of 2.3–2.5 (n=3) compared to medium and IL-2 alone. 37 NK cell clones with yields of &gt; 2E6 cells per clone have been generated using this optimised method and will now be used for Killer Immunoglobulin-like receptor mRNA analyses and antibody based KIR phenotyping (CD158a/h, CD158b, CD158e, CD158i, anti KIR2D). KIR analysed NK cell clones may be used to generate novel highly specific anti KIR antibodies for flowcytometric, quantitative KIR phenotyping. Such a panel can be helpful for optimization of donor selection for allogeneic stem cell transplantation.

Clinical Correlation of Foxp3 Regulatory Gene Expressions and NK Cells in Early Engraftment after Myeloablative Allogeneic Stem Cell Transplantation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2911-2911
Author(s):  
Deok-Hwan Yang ◽  
Chung-Sun Park ◽  
Yeo-Kyeoung Kim ◽  
Je-Jung Lee ◽  
Hyeoung Joon Kim
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Regulatory T Cells ◽  
Stem Cell Transplantation ◽  
Nk Cells ◽  
Cell Transplantation ◽  
Nk Cell ◽  
Gene Expressions ◽  
Foxp3 Gene

Abstract Regulatory T cells (Tregs) have been established as a key role of self tolerance and preventing proliferation of auto- and alloantigen-reactive T cells. CD4+CD25+ Tregs suppress GVHD in animal models, but in human data on GVHD following stem cell transplantation (SCT) is conflicting. We postulate that CD4+Foxp3+Tregs not only reduce the incidence of acute graft-versus host disease (aGVHD) but inhibit the NK cell functions in patients who received allogeneic SCT. CD4+Foxp3+Tregs also may adversely affect the GVL effect and cause the relapse of disease. Patients and Methods: 25 patients (AML:13, ALL:9, CML:2, NHL:1) were undergone allogeneic SCT. Nine patients were infused from unrelated donor and five patients were from HLA one-mismatch. Three patients added alemtuzumab to myeloablative conditioning regimen due to HLA mismatch. Peripheral blood mononuclear cell (PBMC) were separated 3 or 4 weeks after SCT when absolute neutrophil count reached above 1000 × 109/L. NK cells were phenotypically analyzed by flow cytometry using directly conjugated antibodies to CD3 and CD56. CD4+ cells were isolated from PBMC using micro-bead (MACS) and the expression levels of Foxp3 mRNA were assessed by quantitative real-time PCR. Results: 12 patients developed grade 2–4 acute GVHD and 10 patients relapsed. Patients who experienced Gr2-4 aGVHD had significantly lower the relapse rate than those who Gr0-1 aGVHD (P=0.002). Foxp3 gene expressions within CD4+ T cells were significantly lower in Gr2-4 aGVHD patients (median, 4.278 ng/μl) than in Gr0-1 aGVHD patients (median 7.914 ng/μl) except three patients treated with alemtuzumab conditioning (P=0.016). All of three patients used alemtuzumab experienced the relapse and no aGVHD. They also had very lower Foxp3 gene expressions (median, 4.760 ng/μl) than those in Gr0-1 aGVHD patients. Without alemtuzumab used patients, the levels of Foxp3 expression in relapsed patients were significantly higher than those in non-relapsed patients (median, 11.684 ng/μl and 2.031 ng/μl, respectively) (P=0.001). However, we could not find an inverse correlation between NK cell expressions and CD4+Foxp3+Tregs and a positive correlation between infused T cell doses and CD4+Foxp3+Tregs expression. Conclusion: The levels of CD4+Foxp3+Tregs affect the incidence of aGVHD and predict the risk of the relapse. Alemtuzumab may influence the T cell recovery including regulatory T cells after early post-SCT.

scholarly journals Human NK Cells in Autologous Hematopoietic Stem Cell Transplantation for Cancer Treatment

Cancers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1589
Author(s):  
Ane Orrantia ◽  
Iñigo Terrén ◽  
Gabirel Astarloa-Pando ◽  
Olatz Zenarruzabeitia ◽  
Francisco Borrego
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Nk Cells ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Cell Biology ◽  
Nk Cell ◽  
Hematopoietic Stem ◽  
Autologous Hsct

Natural killer (NK) cells are phenotypically and functionally diverse lymphocytes with the ability to recognize and kill malignant cells without prior sensitization, and therefore, they have a relevant role in tumor immunosurveillance. NK cells constitute the main lymphocyte subset in peripheral blood in the first week after hematopoietic stem cell transplantation (HSCT). Although the role that NK cells play in allogenic HSCT settings has been documented for years, their significance and beneficial effects associated with the outcome after autologous HSCT are less recognized. In this review, we have summarized fundamental aspects of NK cell biology, such as, NK cell subset diversity, their effector functions, and differentiation. Moreover, we have reviewed the factors that affect autologous HSCT outcome, with particular attention to the role played by NK cells and their receptor repertoire in this regard.

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