Effect of Plerixafor on Graft Lymphocyte Subsets in NHL Patients Mobilizing Poorly with Chemotherapy Plus G-CSF

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1929-1929
Author(s):  
Esa Jantunen ◽  
Ville Varmavuo ◽  
Taru Kuittinen ◽  
Tapio Nousiainen ◽  
Pentti Mäntymaa
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
Nk Cells ◽  
Lymphocyte Subsets ◽  
Nk Cell ◽  
T Cell Subsets ◽  
Control Group ◽  
High Dose ◽  
Cell Content ◽  
Cd34 Cells

Abstract Abstract 1929 A combination of chemotherapy plus G-CSF (chemomobilization) is commonly used to mobilize CD34+ cells to circulation. Mobilization of CD34+ cells is poor or suboptimal in 20–30 % of patients. Plerixafor, a CXCR4 antagonist, increases the mobilization of CD34+ cells and may also have effect on graft composition subsequently collected. There are no data on lymphocyte subsets in the grafts collected after chemomobilization plus pre-emptively given plerixafor. We have analyzed lymphocyte subsets (CD3, CD4, CD8, NK cells, CD19) in grafts collected on the next morning after plerixafor injection in 13 chemomobilized patients with non-Hodgkin lymphoma. As controls we had the first collections from 13 NHL patients mobilized with chemotherapy plus G-CSF and with yield of 2–6 × 106/kg CD34+ cells with 1–2 aphaereses. The median CD34+ content of the analyzed grafts was 1.45 × 106/kg in the plerixafor group compared to 1.8 × 106/kg in the controls (p=n.s.). The number of T-cell subsets and NK cells were significantly higher in plerixafor mobilized grafts (Table 1). CD19+ B cells were infrequent in both groups.Table 1.Lymphocyte subsets of the grafts.Stem cell collection with plerixafor, median (range)Stem cell collection without plerixafor, median (range)Significance pGraft volume (ml)100 (43–190)80 (45–140)0.280Graft sample preservation time (days)299 (31–450)291 (103–397)0.898CD34+ cell content (x 106/ kg) after 7-AAD1.45 (0.40–4.40)1.80 (0.31–4.74)0.858CD3+ cell content (x 106/kg)75.3 (14.6–327.3)21.3 (9.1–159.4)0.004CD4+ cell content (x 106/kg)32.7 (10.6–132.8)12.4 (6.9–51.5)0.002CD8+ cell content (x 106/kg)33.4 (4.2–200.5)8.8 (2.2–125.0)0.006CD19+ cell content (x 106/kg)0 (0–0)0 (0–0)NANK cell content (x 106/kg)5.1 (0.2–30.40)1.5 (0.3–8.0)0.045CD4+/CD8+ cell ratio0.98 (0.34–3.04)1.41 (0.28–5.06)0.2287-AAD, 7-Aminoactinomycin D; NK, natural killer. All except one patient has received high-dose therapy with blood stem cell support. The median CD34+ cell dose was 3.1 × 106/kg in plerixafor treated group and 3.3 × 106/kg in the control group, respectively. Time to neutrophil engraftment was comparable between the groups. There were two patients in the plerixafor group with late platelet engraftment (1 and 6 months). Addition of plerixafor to chemomobilization in poor mobilizers results in increased content of T lymphocytes and NK cells in the graft but do not appear to mobilize B lymphocytes. Whether higher T cell and NK cell content are associated with more rapid immune reconstitution and survival should be evaluated in larger patient series with longer follow-up. Disclosures: Jantunen: Genzyme: Honoraria. Kuittinen:Roche: Consultancy.

scholarly journals Identification of Lymphocyte Subsets Associated with Outcomes in Patients with Hematological Malignancy Following Allogeneic Stem Cell Transplantation: A Single Institute Study

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2115-2115
Author(s):  
Taiki Ando ◽  
Yasufumi Ishiyama ◽  
Takayoshi Tachibana ◽  
Masatsugu Tanaka ◽  
Heiwa Kanamori ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nk Cells ◽  
Immune Reconstitution ◽  
Lymphocyte Subsets ◽  
Chronic Gvhd ◽  
T Cell Subsets ◽  
Transplant Outcome ◽  
Cell Sources

Abstract Background: Immune reconstitution after allogeneic stem cell transplantation (SCT) is a complicated process influenced by factors such as preconditioning regimens, graft-versus-host disease (GVHD) prophylaxis, and grafts. We studied the association between the kinetics of lymphocyte subsets and transplant outcome to clarify the differences in immune reconstitution after hematopoietic cell transplantation according to stem cell sources and its clinical significance. Patients and Methods: Clinical data were collected from patients' medical charts at Kanagawa Cancer Center, Yokohama, Japan. Patients with hematological malignancies aged ≥18 years old who underwent SCT between April 2009 and December 2017 were initially selected. Those who died or experienced disease relapse before day 100 post SCT were excluded. We measured absolute lymphocyte count (ALC) and lymphocyte subsets by flow cytometry with antibodies against CD2, CD3, CD4, CD8, CD11b, CD11c, CD16, CD25, CD29, CD56, CD57, CD45RA, and CD45RO on days 100, 180, 365, and 730 post SCT. Results: The final cohort included 314 patients (acute leukemia, n = 249; myelodysplastic syndrome, n = 44; chronic myelogenous leukemia, n = 9; malignant lymphoma, n = 6; and others, n = 6). The median age was 51 (range: 18- 69) years, with 184 males and 130 females. The disease risk at transplantation was standard in 209 and high in 105 patients. Myeloablative preconditioning was administered to 114 and reduced-intensity preconditioning to 200 patients. Bone marrow transplantation (BMT), peripheral blood SCT (PBSCT), and cord blood transplantation (CBT) were performed in 121, 57, and 136 patients, respectively. A calcineurin inhibitor with short-term methotrexate was mainly used for GVHD prevention. The median follow-up of surviving patients was 869 (range: 103-3074) days. The 2-year overall survival (OS), cumulative incidence of relapse (CIR), and non-relapse mortality (NRM) in BMT, PBSCT, and CBT were 62%, 68%, and 76% (P = 0.023); 33%, 38%, and 27% (P = 0.068); and 17%, 16%, and 13% (P = 0.82); respectively. The 2-year cumulative incidence of chronic GVHD was 43% in BMT, 45% in PBSCT, and 28% in CBT (P = 0.027). There were significant differences between lymphocyte subset recovery and stem cell sources (Table). ALC; CD20+ B cell; CD4+, CD4+CD29+, CD4+CD45RO+, CD4+CD45RO− , and CD4+CD45RA+ T cell subsets; and CD3−CD56+ and CD16+CD57− natural killer (NK) cell subsets were significantly elevated in CBT compared with BMT and PBSCT at day 100 post SCT. Conversely, CD8+CD11b+ and CD8+CD11b− T cell subsets and CD3+CD56+ NKT cells were significantly lower in CBT than in BMT and PBSCT on day 100. Univariate analysis revealed that lymphocyte subsets exhibiting higher levels of CD20+ B cell; CD16+CD57− and CD3−CD56+ NK cells; and CD4+CD25+, CD4+CD29+, CD4+CD45RA+, CD8+CD11b+, and CD8+CD11b− T cell subsets at day 100 were associated with a better 2-year OS. There were strong correlations with a lower CIR and higher CD16+CD57+ and CD16+CD57− NK cell levels . A higher incidence of chronic GVHD was associated with lower levels of CD16+CD57− NK cells and CD4+CD25+ T cells and with higher levels of CD8+CD11b+ T cells and CD8+CD11b− and CD3+CD56+ NKT cells. Further, a lower NRM correlated with higher levels of CD20+ B cells and CD8+CD11b− T cells. The lymphocyte subsets were used for multivariate analysis. Favorable factors for better OS were higher levels of CD16+CD57− NK cells [hazard ratio (HR), 0.62; 95% confidence index (CI), 0.38-0.81; P = 0.024] and CD20+ B cells (HR, 0.56; 95% CI, 0.31-0.0.98; P = 0.048). Prognostic factors for lower CIR were higher levels of CD16+CD57+ NK cells (HR, 0.51; 95% CI, 0.27-0.95; P = 0.034) and CD16+CD57− NK cells (HR, 0.52; 95% CI, 0.28-0.99; P = 0.048). A lower incidence of NRM was associated with higher levels of CD8+CD11b− T cells (HR, 0.18; 95% CI, 0.08-0.39; P < 0.001) and CD20+ B cells (HR, 0.24; 95% CI, 0.08-0.70; P = 0.0088). High levels of CD8+CD11b+ T cells were an independent predictor for a higher incidence of chronic GVHD (HR, 2.38; 95% CI, 1.22-4.95; P = 0.012). Conclusions: The distinct differences in immune reconstitution according to stem cell sources and lymphocyte subset analysis at day 100 post SCT are useful for predicting transplant outcome. Furthermore, the results suggest that characteristic immune recovery in CBT positively affects transplant outcome. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Naive NK Cell Repertoire in the Circulation of Haploidentical Stem Cell Donors Pre Mobilization Predicts Rejection of Cord Myeloid Cells in Patients Undergoing Combined Haploidentical and Unrelated Cord Blood HSCT

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2199-2199
Author(s):  
Mattias Carlsten ◽  
Robert N. Reger ◽  
Ritesh Kotecha ◽  
Enkhtsetseg Purev ◽  
Xin Tian ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Nk Cells ◽  
Nk Cell ◽  
Stem Cell Mobilization ◽  
Cell Repertoire ◽  
Post Transplant ◽  
Cd34 Cells ◽  
Cell Mobilization ◽  
Unrelated Cord Blood

Abstract Background: For patients (pts) with severe aplastic anemia (SAA) lacking an HLA identical donor, outcomes of hematopoietic stem cell transplantation (HSCT) using unrelated cord blood (UCB) units or haplo-identical donors (HDs) have historically been associated with high graft failure rates and poor survival. In an ongoing clinical trial at the NHLBI, we have observed excellent engraftment (100%) and survival (91%) in SAA pts (n=27) receiving a transplant that co-infuses a single UCB unit with CD34-selectedCD3-depletedcells from a haplo-identical relative. Although cord myeloid engraftment(defined as cord ANC >500/μL) occurred at<day 100 in the majority of pts, a significant fraction of pts had delayed (>day 100) or no cord myeloid engraftment. In this analysis, we investigated factors that may have impeded cord myeloid engraftment following UCB/HD transplantation. Methods: Flow-based NK cell phenotyping using a BD Fortessa II instrument was performed on blood obtained pre-transplant from HDs used for the first 18 SAA pts undergoing UCB/HD transplantation. Lineage specific chimerism was measured by PCR of microsatellites (PowerPlex 16 HS Systemkit/Promega) using DNA from flow sorted cells (BD FACSAria) collected multiple time points post-transplant.KIR-ligand incompatibility in the HD vs UCB directionwas defined using high-resolution HLA typing. Results: 13/18 (72%) pts had cord myeloid engraftment before day 100 while 5/18 (28%) had delayed or no cord myeloid engraftment. Remarkably, delayed or no cord myeloid engraftment occurred exclusively in pts transplanted with KIR-ligand incompatibility in the HD vs UCB direction (n=9) (Figure 1A). In contrast, all 9 pts transplanted with KIR-ligand compatibility in the HD vs UCB direction achieved cord myeloid engraftment by ²day 48 (median day 35) post-transplant. Chimerism analysis performed on blood obtained 30+ days post-transplant revealed NK cell chimerism was ³ 90% cord in origin in all 9 pts transplanted with KIR-ligand compatible grafts. In contrast, amongst the 9 pts receiving a KIR-ligand incompatible transplant, NK cell chimerism was predominantly HD in origin with only a minor fraction of cord NK cells detected 30-200 days post-transplant (Figure 1B). Predominant HD NK cell chimerism in pts receiving a KIR-ligand incompatible transplant was associated with lower degrees of cord myeloid chimerism compared to KIR-ligand compatible recipients. Further analysis of the KIR-ligand incompatible cohort revealed distinct heterogeneity in the time to cord myeloid engraftment (Figure 1A). Although delayed or no cord myeloid engraftment was observed in 5/9 recipients of KIR-ligand incompatible transplants, 4/9 pts in this cohort had cord engraftment at a similar time as pts transplanted with KIR-ligand compatible grafts (median 35 vs. 35 days). This variability in time to cord myeloid engraftment was not associated with stem cell dose, degree of HD NK cell chimerism, type of KIR-ligand incompatibility or KIR haplotype. However, we observed a strong correlation between the proportion of naive NK cells in circulation of HDs before stem cell mobilization with delayed or no myeloid cord engraftment (Figure 1C). With the exception of one patient who had failed HD engraftment, only transplants of CD34+ cells from HDs with a predominantly naive NK cell repertoire, expressing high frequencies of the NKG2A receptor concomitant with low frequencies of NKG2C, Lir-1 and CD57 resulted in delayed or no cord myeloid engraftment (p<0.05). Conclusions: Our study provides the first evidence that NK cells from engrafting CD34+ cells from selected HDs can significantly delay or completely inhibit cord myeloid engraftment following UCB/HD transplantation. Suppression of cord hematopoiesis appears to be restricted to NK cells originating from HDs withHD vs UCB KIR-ligand incompatibility who have a large naive NK cell repertoire in their circulation prior to stem cell mobilization. The myelosuppressive effects of these NK cells are consistent with recentlypublished data showing a naive NK cell repertoire in stem cell donors predicts a reduced risk of AML relapse post-allogeneic HSCT.Further studies defining the mechanisms through which naive NK cells suppress cord hematopoiesis followingUCB/HDtransplantation could shed insights into methods to optimize NK cell mediated graft-vs-leukemia followingallogeneicHSCT of myeloid leukemias. Disclosures No relevant conflicts of interest to declare.

scholarly journals Characterization of the DNAM-1, TIGIT and TACTILE Axis on Circulating NK, NKT-Like and T Cell Subsets in Patients with Acute Myeloid Leukemia

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2171
Author(s):  
Isabel Valhondo ◽  
Fakhri Hassouneh ◽  
Nelson Lopez-Sejas ◽  
Alejandra Pera ◽  
Beatriz Sanchez-Correa ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Healthy Volunteers ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Cell Activation ◽  
Nk Cell ◽  
T Cell Subsets ◽  
Acute Myeloid

Background: Acute myeloid leukemia (AML) remains a major clinical challenge due to poor overall survival, which is even more dramatic in elderly patients. TIGIT, an inhibitory receptor that interacts with CD155 and CD112 molecules, is considered as a checkpoint in T and NK cell activation. This receptor shares ligands with the co-stimulatory receptor DNAM-1 and with TACTILE. The aim of this work was to analyze the expression of DNAM-1, TIGIT and TACTILE in NK cells and T cell subsets in AML patients. Methods: We have studied 36 patients at the time of diagnosis of AML and 20 healthy volunteers. The expression of DNAM-1, TIGIT and TACTILE in NK cells and T cells, according to the expression of CD3 and CD56, was performed by flow cytometry. Results: NK cells, CD56− T cells and CD56+ T (NKT-like) cells from AML patients presented a reduced expression of DNAM-1 compared with healthy volunteers. An increased expression of TIGIT was observed in mainstream CD56− T cells. No differences were observed in the expression of TACTILE. Simplified presentation of incredibly complex evaluations (SPICE) analysis of the co-expression of DNAM-1, TIGIT and TACTILE showed an increase in NK and T cells lacking DNAM-1 and co-expressing TIGIT and TACTILE. Low percentages of DNAM-1−TIGIT+TACTILE+ NK cells and DNAM-1− TIGIT+TACTILE+ CD56− T cells were associated with a better survival of AML patients. Conclusions: The expression of DNAM-1 is reduced in NK cells and in CD4+ and CD8+ T cells from AML patients compared with those from healthy volunteers. An increased percentage of NK and T cells lacking DNAM-1 and co-expressing TIGIT and TACTILE is associated with patient survival, supporting the role of TIGIT as a novel candidate for checkpoint blockade.

Platelet Engraftment (PE) in AML Patients Receiving HLA Matched Sibling Donor Allogeneic Bone Marrow Transplant (ABMT) Correlates with Inhibitory HLA Ligand Groups for Killer Immunoglobulin-Like Receptors (KIRs).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 836-836
Author(s):  
Ronald Sobecks ◽  
Edward J. Ball ◽  
Lisa Rybicki ◽  
Stacey Brown ◽  
Jaroslaw Maciejewski ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
Nk Cells ◽  
Median Time ◽  
Nk Cell ◽  
Multivariable Analysis ◽  
Sibling Donor ◽  
Cell Dose ◽  
Hla Ligands

Abstract The interaction of KIRs with target cell HLA class I molecules regulates the activity of NK cells and some T cell populations. KIR interactions are reported to influence allogeneic hematopoietic stem cell transplant outcomes, particularly for AML. We have previously reported that AML pts homozygous for C1 or C2 have superior survival and lower relapse rates than do pts heterozygous for C1/C2. However, the influence of KIR matching on PE has not been well described. We therefore analyzed the KIR ligand phenotypes of 60 AML pts who received HLA identical sibling donor myeloablative ABMT from 4/9/97 – 11/5/03. The median age was 45 years (range, 8–62 yrs). At transplant a minority (40%) were in CR. All pts received a busulfan/cyclophosphamide based preparative regimen and T-cell replete bone marrow as their stem cell source. Patient HLA KIR ligands were categorized as: 1) HLA-Cw group C1- or C2 - homozygous vs C1/C2 heterozygous; and 2) HLA-Bw4 (positive or negative) (reviewed in Farag et al Blood100:1035, 2002). Kaplan Meier estimates of median time to PE &gt;20 K/μL and &gt;50 K/μL were 23 and 30 days, respectively. PE was next assessed in relation to the inhibitory HLA KIR ligand group expressed. PE &gt;20 K/μL was superior for those C1 or C2 homozygous (n=26) compared to C1/C2 heterozygotes (n=34) (median 21 vs 26 days, p=0.049) and 31 Bw4 negative pts had superior PE compared to 29 Bw4+ pts (median 21 vs 30 days, p=0.012); these findings remained significant in multivariable analysis. A similar analysis performed for PE &gt;50 K/μL found that Bw4 negative pts had superior PE compared to Bw4 + pts (median 26 vs 38 days, p=0.015); this remained significant in multivariable analysis. 57 cases had KIR genotyping performed for those KIRs with established HLA ligands and there were no cases in which the donor did not have at least one inhibitory KIR gene specific for expressed HLA ligands. Age at transplant, number of prior chemotherapy regimens, disease status at transplant and CMV status were not predictive of PE. Since both C1/C2 heterozygosity and Bw4+ status correlated with reduced early PE, the possible interaction of these two variables was next investigated. The analysis for PE &gt;20 K/μL and &gt;50 K/μL suggested an additive effect: pts lacking expression of both these variables had the most rapid PE, while those who expressed both variables had the slowest PE. Those who expressed only 1 of the variables had an intermediate time to PE as shown below: HLA KIR Ligand Status N Median CD34+ cell dose (x10^6/kg) Median Total Nucleated Cell Dose (x10^8/kg) Median time to PE&gt;20K/μL Median time to PE &gt;50K/μL C1/C2 and Bw4 negative 14 2.02 2.64 19 days 25 days C1/C2 positive and Bw4 negative OR C1/C2 negative and Bw4 positive 29 1.92 2.62 23 days 29 days C1/C2 and Bw4 positive 17 1.87 2.60 31 days 41 days p= 0.54 p=0.82 p=0.003 p=0.003 These results may suggest that donor NK cells control host effector cells that delay PE. When minimal opportunity for inhibitory KIR engagement exists (C1/C2 negative, Bw4 negative) maximal NK cell control is expected and rapid PE ensues. When maximal opportunity for inhibitory KIR engagement exists (C1/C2 positive, Bw4 positive) donor NK cell controlling activity would be reduced, leading to delayed PE.

Natural Killer Cell Immune Reconstitution Predicts Outcomes for Patients with Chronic Lymphocytic Leukemia Undergoing Allogeneic Stem Cell Transplantation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3300-3300
Author(s):  
Don Benson ◽  
Leslie Andritsos ◽  
Mehdi Hamadani ◽  
Thomas Lin ◽  
Joseph Flynn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Immune Reconstitution ◽  
Nk Cell ◽  
Disease Status ◽  
Lymphocytic Leukemia ◽  
T Cell Subsets ◽  
Cell Recovery

Abstract Introduction: Chronic lymphocytic leukemia (CLL), the most common form of leukemia in the Western hemisphere, is associated with severe innate, adaptive and humoral immune dysregulation. CLL remains essentially incurable, with the potential exception of allogeneic stem cell transplantation (ASCT). Natural killer (NK) cells are CD56(+), CD3(−) large granular lymphocytes that comprise a key cellular subset of the innate immune system. Preliminary in vitro data suggest an NK cell versus CLL effect exists, similar to that observed in acute myeloid leukemia (AML) and other blood cancers. Novel immune therapies for CLL (e.g., rituximab, alemtuzumab) likely exert anti-tumor effect, in part, through NK cells, in fact. Although NK cells contribute to the graft-versus-tumor effect following ASCT for other blood cancers, little is known regarding the potential role NK cells may play in the clinical allogeneic transplant setting for CLL. Herein, we provide, to our knowledge, the first report regarding NK cell immune reconstitution following ASCT for CLL. Methods: 27 CLL patients underwent reduced intensity conditioning (RIC) with ASCT. Median age was 52 years (43–69), median number of prior therapies was 3 (2–11). 55% had chemotherapy-refractory disease, and 55% had “high-risk” cytogenetics by FISH (deletion 17p or 11q22-23 abnormality). 14 patients had sibling donors, 15 had volunteerunrelated donors. Conditioning regimens included Fludarabine/TBI/Alemtuzumab (n=8), Fludarabine/Busulfan with (n=9) or without ATG (n=6), and Fludarabine/Cyclophosphamide (n=4). GVHD prophylaxis consisted of tacrolimus/MMF (n=8) or tacrolimus/methotrexate (n=19). Patients underwent bone marrow assessment prior to day +75 following ASCT. Marrow was studied for engraftment, donor chimerism, and disease status as well as lymphoid immune reconstitution by percentage of total lymphocytes and absolute lymphocyte counts by multi-color flow cytometry. Results: NK cell immune reconstitution was predicted by disease status at transplantation. Patients in complete or partial remission at the time of ASCT had more robust NK cell recovery (mean = 45% of total lymphocytes +/− SEM 5%) as compared to patients entering transplant with refractory disease (16% +/− 1, p < 0.01). No differences were observed in CD4(+) or CD8(+) T cells and no lymphocyte subset recovery was associated with CD34(+) or CD3(+) cell dosage. Achieving complete donor chimerism by day +60 was associated with robust NK cell recovery (55% +/− 1 versus 7% +/−1, p = 0.02), recovery of CD4 and CD8 T cells was not associated with chimerism status, however. Patients who went onto exhibit a complete response to ASCT had greater early NK cell reconstitution (31% +/− 3) as compared to those who had no response (8% +/− 1, p = 0.01). No differences in T cell subsets were associated with response. Patients who ultimately achieved complete remission following transplant had a lower CLL:NK cell ratio in marrow (0.35 +/− 0.07) than those who did not (8.1 +/− 1, p = 0.01). However, differences in CLL:CD4(+) and CLL:CD8(+) T cells were not predictive of response. Trends to improvement in progression free survival and overall survival were observed for patients with NK cell reconstitution above the median for the group as compared to those below; no such trends were observed regarding T cell subsets. Greater NK cell reconstitution trended towards ultimate eradication of minimal residual disease following ASCT, but no such trends were observed for T cell subsets. Conclusions: Early NK cell recovery predicts survival following autologous and allogeneic SCT in a number of hematologic malignancies; however, little is known regarding this phenomenon in CLL. To our knowledge, these are the first findings to implicate a potentially important therapeutic role for early NK cell compartment recovery in CLL following ASCT. Further research into restoring and augmenting NK cell function following RIC/ASCT for CLL is warranted.

scholarly journals The number of CD56dim NK cells in the graft has a major impact on risk of disease relapse following allo-HSCT

2017 ◽  
Vol 1 (19) ◽  
pp. 1589-1597 ◽  
Author(s):  
Luke Maggs ◽  
Francesca Kinsella ◽  
Y. L. Tracey Chan ◽  
Suzy Eldershaw ◽  
Duncan Murray ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
Nk Cells ◽  
Clinical Outcomes ◽  
Nk Cell ◽  
Disease Relapse ◽  
Cell Dose ◽  
Key Points ◽  
Stem Cell Graft ◽  
Risk Of Disease

Key Points A stem cell graft NK cell dose below 6.3 × 106 cells per kg associates with risk of disease relapse following T-cell–depleted allo-HSCT. Clinical outcomes of patients undergoing allo-HSCT may be improved by setting an NK cell threshold within donor stem cell grafts.

Preclinical Ex-Vivo Generation of Clinical Relevant Amounts of NK Cells from CD34+ Progenitor Cells for Immunotherapy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2916-2916
Author(s):  
Jan Spanholtz ◽  
T. M. de Witte ◽  
Marleen Tordoir ◽  
Harry Dolstra
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Nk Cells ◽  
Progenitor Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Cell Product ◽  
Cytotoxicity Assays ◽  
Cd34 Cells ◽  
Specific Antigens

Abstract Alloreactive donor Natural Killer (NK) cells, displaying a KIR-ligand mismatch with the recipient play a pivotal role in graft-versus-leukemia (GVL) reactivity without significant graft-versus-host disease (GVHD) following haploidentical stem cell transplantation. Therefore, infusions of haploidentical NK cells are suggested to become an attractive approach for cancer immunotherapy. So far, difficulties in isolation and expansion of peripheral NK cells resulted in only limited data about safety and clinical efficacy of purified NK cell infusions. Therefore, we have developed a novel culture system without the use of feeder cells for the ex vivo generation of NK cells from CD34+ hematopoietic progenitor cells (HPCs) isolated from cord blood (CB) or bone marrow (BM). It is based on a two-step procedure using an expansion and a differentiation step. The NK cell generation system uses mainly cytokines such as SCF, TPO, Flt3-L, IL-2, IL-7 and IL-15 and specific modified glycosaminoglycans (GAGs) to direct and control the two phases. The developmental phase and the final NK cell product is controlled and characterized by immunophenotyping using multi-colour flow cytometry and CFSE-based cytotoxicity assays against various tumor cells. Our system generates a homogeneous final cell product of CD56+/CD3- cells with a purity of &gt;99%. A total cell expansion of more than 5×10^4 fold allows to generate 5×10^10 NK cells from 1×10^6 CB CD34+ stem and progenitor cells within 4–5 weeks of culture. For BM cells an expansion rate of more than 1×10^4 fold was detected after a 5–6 week cell culture period. During the two week expansion phase step,we expand UCB CD34+ cells more than 100 fold. Phenotypic analysis showed a decrease of stem cell-specific antigens such as CD34 and CD117 during the first three weeks, whereas antigens specific for NK cell progenitors and mature NK cells such as CD56, CD94 and CD161 are up-regulated after initiating differentiation at day 14. Furthermore, distinct cell populations can be detected reflecting NK cell developmental stages in vitro. Effective differentiation of the expanded progenitor cells into mature NK cells is characterized by the expression of NK cell-specific antigens including CD56, CD94, NKG2A, NKG2D and NCRs as well as homing receptors such as CD62L, CXCR4 and CCR7. The final NK cell product shows high expression levels of inhibitory and activating receptors as well the intrinsic capability to express KIR, which is detected by flow cytometry after 6–7 weeks of culture. Cytotoxicity assays demonstrated robust lysis of more than 90% against AML as well as melanoma tumor cell lines. This system, with its huge expansion potential to generate highly activated NK cells with homing capability, is the basis for a first clinical trial in 2009, to infuse haploidentical NK cells generated from CD34+ cells in poor-risk AML patients. The use of our defined culture conditions enables new prospects in NK cell research, regarding NK cell development and NK cell maturation, as well as new aspects for the clinical use of NK cell products derived from HPCs.

Expansion of Donor NK Cells for Adoptive Immunotherapy in Haploidentical Stem Cell Transplantation: A Phase I–II Study

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3893-3893
Author(s):  
Uwe Siegler ◽  
Sandrine Meyer-Monard ◽  
Simon Jörger ◽  
Martin Stern ◽  
Andre Tichelli ◽  
...  
Keyword(s):  
T Cell ◽  
Phase I ◽  
Nk Cells ◽  
Large Scale ◽  
Nk Cell ◽  
Human Cancer ◽  
Hla Class I ◽  
Cytolytic Activity ◽  
Cell Content ◽  
Target Ratio

Abstract Natural killer (NK) lymphocytes are essential for anti-cancer defense. Transplantations of haploidentical hematopoietic stem cells (HSCT) across HLA class I barriers highlighted the graft-versus-leukemia effect of alloreactive NK cells and strengthened prospects for NK cell immunotherapy in human cancer. Reconstituting NK cells remain immature with impaired cytotoxicity for more than 6 months after HSCT, suggesting a benefit of adoptive transfer of mature NK cells to provide the patient with competent cytotoxic effectors. We previously reported the feasibility of purifying NK cells in numbers sufficient for infusions of 1×107 cells/kg (Passweg et al. 2004). Here we describe an approach of increasing the effector to target ratio by large-scale good manufacturing practice (GMP)-compliant expansion of NK cells and preemptive multiple donor lymphocyte infusions (NK-DLI) after haploidentical HSCT for hematological malignancies. This single centre phase I–II clinical study was approved by the ethical committee. NK cells were purified from a median of 7.4L (range: 5.6–10.0L) unstimulated leukapheresis of 5 healthy donors by T cell depletion and subsequent NK cell selection with anti-CD3 and anti-CD56 coated immunomagnetic microbeads on the CliniMACS_ device. A median of 5.32×108 (2.87–6.42×108) CD56+CD3- NK cells was obtained with a purity of 98.5% (71.9–99.6%), and a residual CD3+ T cell content of 0.030% (<0.001–0.070%) corresponding to a T cell depletion efficiency of 3.51 log (3.15–4.87 log). NK cells were cultured in 30 air-permeable bags and up to 7.8L of GMP-certified medium containing human serum, IL-2, anti-CD3 monoclonal antibody OKT-3 and irradiated autologous feeder cells. After 15–20 days of culture, NK cell numbers increased on average 50 fold (12–137 fold). CD3+ T cells concomitantly expanded to 0.59% (0.014–1.68%) of total cells. A second CD3 depletion was performed with 3 NK cell products which exceeded the maximal T cell dose of 0.5×105/kg as required by the planned therapeutic study protocol. T cell content was reduced by 1.07 log (0.84–3.17 log) at the expense of a significant NK cell loss of 47% (32–76%). Bacterial contamination tests from all culture bags were negative. These results demonstrate the feasibility of clinical grade large-scale ex vivo NK cell expansion. GMP-expanded NK cells exhibited high levels of activating receptors NKG2D and NKp44 (3.1 and 16.8 fold increase above basal levels). The proportions of alloreactive NK cells with single killer immunoglobulin-like receptor (KIR) specificities remained stable comprising 2.4–16.0% and 5.2–14.2% of cells before and after expansion. GMP-expanded NK cells were more cytotoxic against K562 target cells than freshly isolated NK cells (mean±SEM: 61.0±3.1% vs. 27.0±8.0% specific lysis at 10:1 effector to target ratio). The cytolytic activity against KIR-HLA class I mismatched primary AML blasts was 7.0±3.7%, reflecting the content of alloreactive NK cell subsets. This is supported by our finding that purified single-KIR alloreactive NK cells lysed mismatched AML blasts with a high efficiency (22.0±6.3% at 10:1 ratio), indicating the anti-leukemic capacity of GMP-expanded NK cells. Two NK-DLI products with a total of 21.0×108 and 189.4×108 NK cells were generated for two patients and divided into three escalating doses (1×106, 1×107 and 1.5×107/kg) for patient 1 and into four doses (1×106, 1×107 and twice 1×108/kg) for patient 2. The infusions were tolerated without any acute adverse effects. In conclusion, we established and clinically implemented a protocol suitable for GMPcompliant expansion of cytokine-activated NK cells allowing multiple infusions of high numbers of cells with strong cytolytic activity. These results provide the basis for a prospective clinical efficacy trial to advance the therapeutic field of NK-DLI against human cancer.

scholarly journals Comparative effects of six probiotic strains on immune functionin vitro

2011 ◽  
Vol 108 (3) ◽  
pp. 459-470 ◽  
Author(s):  
Honglin Dong ◽  
Ian Rowland ◽  
Parveen Yaqoob
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Bifidobacterium Longum ◽  
Cell Activity ◽  
T Cell Subsets ◽  
Nk Cell Activity ◽  
Tnf Α ◽  
Significant Difference ◽  
Probiotic Strains

There is considerable interest in the strain specificity of immune modulation by probiotics. The present study compared the immunomodulatory properties of six probiotic strains of different species and two genera in a human peripheral blood mononuclear cell (PBMC) modelin vitro. Live cells of lactobacilli (Lactobacillus caseiShirota,L. rhamnosusGG,L. plantarumNCIMB 8826 andL. reuteriNCIMB 11951) and bifidobacteria (Bifidobacterium longumSP 07/3 andB. bifidumMF 20/5) were individually incubated with PBMC from seven healthy subjects for 24 h. Probiotic strains increased the proportion of CD69+on lymphocytes, T cells, T cell subsets and natural killer (NK) cells, and increased the proportion of CD25+, mainly on lymphocytes and NK cells. The effects on activation marker expression did not appear to be strain specific. NK cell activity was significantly increased by all six strains, without any significant difference between strains. Probiotic strains increased production of IL-1β, IL-6, IL-10, TNF-α, granulocyte-macrophage colony-stimulating factor and macrophage inflammatory protein 1α to different extents, but had no effect on the production of IL-2, IL-4, IL-5 or TNF-β. The cytokines that showed strain-specific modulation included IL-10, interferon-γ, TNF-α, IL-12p70, IL-6 and monocyte chemotactic protein-1. TheLactobacillusstrains tended to promote T helper 1 cytokines, whereas bifidobacterial strains tended to produce a more anti-inflammatory profile. The results suggest that there was limited evidence of strain-specific effects of probiotics with respect to T cell and NK cell activation or NK cell activity, whereas production of some cytokines was differentially influenced by probiotic strains.

scholarly journals Human natural killer cell committed thymocytes and their relation to the T cell lineage.

1993 ◽  
Vol 178 (6) ◽  
pp. 1857-1866 ◽  
Author(s):  
M J Sánchez ◽  
H Spits ◽  
L L Lanier ◽  
J H Phillips
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Cell Lineage ◽  
Surface Expression ◽  
Cd34 Cells ◽  
Clonogenic Potential ◽  
Antigenic Phenotype

Recent studies have demonstrated that mature natural killer (NK) cells can be grown from human triple negative (TN; CD3-, CD4-, CD8-) thymocytes, suggesting that a common NK/T cell precursor exists within the thymus that can give rise to both NK cells and T cells under appropriate conditions. In the present study, we have investigated human fetal and postnatal thymus to determine whether NK cells and their precursors exist within this tissue and whether NK cells can be distinguished from T cell progenitors. Based on the surface expression of CD56 (an NK cell-associated antigen) and CD5 (a T cell-associated antigen), three phenotypically distinctive populations of TN thymocytes were identified. CD56+, CD5-; CD56-, CD5-, and CD56-, CD5+. The CD56+, CD5- population of TN thymocytes, although displaying a low cytolytic function against NK sensitive tumor cell targets, were similar in antigenic phenotype to fetal liver NK cells, gave rise to NK cell clones, and were unable to generate T cells in mouse fetal thymic organ cultures (mFTOC). This population of thymocytes represents a relatively mature population of lineage-committed NK cells. The CD56-, CD5- population of TN thymocytes were similar to thymic NK cells in antigenic phenotype and NK cell clonogenic potential. Clones derived from this population of TN thymocytes acquired CD56 surface expression and NK cell cytolytic function. CD56-, CD5- TN thymocytes thus contain a novel population of NK cell-committed precursors. The CD56-, CD5- population of TN thymocytes also contains a small percentage of CD34+ cells, which demonstrate no in vitro clonogenic potential, but possess T cell reconstituting capabilities in mFTOC. The majority of TN thymocytes do not express CD56, but coexpress CD34 and CD5. These CD56-, CD5+, CD34+ cells demonstrate no NK or T cell clonogenic potential, but are extremely efficient in repopulating mFTOC and differentiating into CD3+, CD4+, CD8+ T cells. The results of this investigation have identified NK cells and NK cell precursors in the human thymus and have shown that these cell types are unable to differentiate along the T cell lineage pathway. Thus, while a common NK/T cell progenitor likely exists, once committed to the NK cell lineage these cells no longer have the capacity to develop along the T cell developmental pathway.

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