Clinical Grade Isolation of Regulatory T Cells From G-CSF Mobilized PBSC Improves with Initial Depletion of Monocytes.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1176-1176
Author(s):  
Dolores Mahmud ◽  
Youngmin Park ◽  
Nadim Mahmud ◽  
Damiano Rondelli
Keyword(s):  
Stem Cells ◽  
Clinical Trials ◽  
T Cells ◽  
Positive Selection ◽  
Peripheral Blood ◽  
Negative Selection ◽  
Clinical Grade ◽  
Step Process ◽  
Cd34 Cells ◽  
Selection Of

Abstract Abstract 1176 We have recently demonstrated that G-CSF mobilized peripheral blood stem cells (PBSC) CD4+CD25+FoxP3+ cells (Tregs) prevent anti-CD34+ hematopoietic stem cells T cell alloreactivity in-vitro and co-transplantation of CD34+ cells and Tregs does not affect human stem cell engraftment in NOD/SCID mice (Mahmud D et al. Biol Blood Marrow Transplant, 2010). Since only a small number of Tregs can be isolated from normal peripheral blood we examined whether PBSC can be a useful source of Tregs for future clinical trials. Five leukapheresis products from healthy donors who received rh G-CSF at 10 ug/kg daily for 5 days were processed using the CliniMACS instrument (Miltenyi Biotec, Auburn, CA). CliniMACS CD34 reagent was initially used to isolate CD34+ cells. To isolate Tregs a 2-step procedure was initially utilized. A cocktail of clinical grade CD14, CD8 and CD19 reagents was mixed with the CD34- cells and depletion of monocytes, cytotoxic T cells and B cells was obtained by using the Depletion 2.1 program. The CD4+ cells were then enriched in Tregs by positive selection of CD25+ cells using a clinical grade CD25 reagent (Miltenyi). Because PBSC contain large amount of myeloid cells, and particularly monocytes, this clinical scale 2-step strategy was compared with a 3-step method that included an initial negative selection of CD14+ monocytes, followed by negative selection of CD8+ and CD19+ cells and a positive selection of CD25+ cells. Prior to isolation, the average proportion of CD4+CD25+ cells in PBSC was 0.77±0.26% in 5 separate PBSC products. After the 2-step process the proportion of CD4+CD25+ cells was 35±33% (n=3) vs 72±1% after the 3-step process. Therefore, utilizing the 3-step approach a better yield of Tregs was observed (10 vs 60%). Intracellular expression of FoxP3 was on average 74% in CD4+CD25+ cells obtained with a 3-step process. Contamination of different cell subsets in the final products enriched in Tregs was largely superior following the 2-step as compared to 3-step isolation method. Contaminating monocytes were, on average, 43 vs 5.7%, and contaminating CD8 and CD19+ cells were 12 vs 1.7% and 0.9 vs 0.3%, respectively. In the two procedures using a 3-step approach the final absolute number of Tregs isolated from products containing on average 30 × 109 mononuclear cells, was 95 and 93 × 106, respectively. These findings obtained using clinically available reagents and device, suggest that depletion of monocytes may improve the purity of Treg cell population isolated from PBSC. PBSC may represent a valuable source of Tregs for future clinical trials. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Generation of clinical-grade CD19-specific CAR-modified CD8+ memory stem cells for the treatment of human B-cell malignancies

Blood ◽  
2016 ◽  
Vol 128 (4) ◽  
pp. 519-528 ◽  
Author(s):  
Marianna Sabatino ◽  
Jinhui Hu ◽  
Michele Sommariva ◽  
Sanjivan Gautam ◽  
Vicki Fellowes ◽  
...  
Keyword(s):  
Stem Cells ◽  
Clinical Trials ◽  
T Cells ◽  
B Cell ◽  
Clinical Grade ◽  
B Cell Malignancies ◽  
Car T Cells ◽  
Key Points ◽  
Car T ◽  
Human B Cell

Key Points A platform for the generation of clinical-grade CD19-CAR–modified TSCM. CD19-CAR–modified TSCM mediate superior antitumor responses compared with CD19-CAR T cells currently used in clinical trials.

CD8 is needed for positive selection but differentially required for negative selection of T cells during thymic ontogeny

1993 ◽  
Vol 23 (1) ◽  
pp. 212-216 ◽  
Author(s):  
Wai-Ping Fung-Leung ◽  
Valerie A. Wallace ◽  
Dawn Gray ◽  
William C. Sha ◽  
Hanspeter Pircher ◽  
...  
Keyword(s):  
T Cells ◽  
Positive Selection ◽  
Negative Selection ◽  
Selection Of

Mobilization, Harvesting and Selection of Peripheral Blood Stem Cells in Patients with Autoimmune Diseases Undergoing Non-Myeloablative Autologous Hematopoietic Stem Cell Transplantation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1980-1980
Author(s):  
Laisvyde Statkute ◽  
Larissa Verda ◽  
Yu Oyama ◽  
Marcelo Villa ◽  
Thomas Shook ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Autoimmune Diseases ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Positive Correlation ◽  
Cd34 Cells ◽  
The Mean ◽  
Stem Cell Collection ◽  
Selection Of

Abstract We have analyzed peripheral blood stem cell (PBSC) mobilization, harvesting and selection properties in 128 patients with severe autoimmune diseases undergoing non-myeloablative autologous hematopoietic stem cell transplantation (HSCT) (50 patients with systemic lupus erythematosus (SLE), 43 - with multiple sclerosis (MS), 15 - with Crohn’s disease (CD), 8 - with scleroderma (Scl), and 12 - with others). Female/male ratio and mean age (range) were 90/38 patients, and 34 (14 to 59) years old, respectively. Mobilization regimen included cyclophosphamide 2g/m2 and G-CSF 10 mcg/kg (except for SLE patients 5 mcg/kg). Forty one patients underwent stem cell collection using Baxter CS300, 78 patients - Spectra, and for 9 patients both apheresis machines were utilized. The mean number of aphereses was 1.8 (range 1–10). Patients with SLE required the largest number of apheresis sessions (mean 2.4), comparing to patients with CD (mean 1.9), Scl (mean 1.4), MS (mean 1.3). Five patients additionally required bone marrow harvest for collection of adequate numbers of stem cells. One patient failed to reach CD34+ cell number of 1.0x106/kg, therefore did not proceed to HSCT. The mean number of CD34+ cells in each apheresis unit was 6.07+−6.96x106/kg (the highest of 9.22+−8.52x106/kg in patients with MS, and the lowest of 3.93+−4.48x106/kg in patients with SLE). Ninety eight patients underwent stem cell selection with CEPRATE SC (N=18), Isolex 300iv1.12 (N=2) or Isolex 300iv2.5 (N=78) stem cell concentrator. The mean purity of selected products was 74.3% (the highest of 81.1% attained in patients with Scl); mean recovery of CD34+cells was 61.2%. T cell reduction by average of 3.7 logs was achieved. The mean number of infused CD34+ cells was 7.24+−5.5x106/kg. The highest mean number of CD34+ cells/kg were infused to patients with MS (9.04+−6.74x106/kg), the lowest - to patients with SLE (5.78+−4.13x106/kg). We found a moderate positive correlation between peripheral blood (PB) CD34+ cells/ul and PB WBC/ul (R=0.34, p<0.05), PB platelets/ul (R=0.51, p<0.05) and a strong positive correlation between PB CD34+ cells/ul and the number of CD34+ cells/kg/apheresis (R=0.67, p<0.05). A weak positive correlation was observed between the number of infused CD34+cells/kg and faster WBC engraftment (ANC>500) and platelet engraftment (platelet count>20K). There was no toxicity observed in our patient population during peri-mobilization period except for 1 patient with SLE who died of disseminated mucormycosis 1 week after stem cell collection. Mobilization and selection of PBSC are safe and efficient in patients with severe autoimmune diseases undergoing non-myeloablative autologous HSCT.

Human IPS Cells Generated From Adult Peripheral Blood Cells and Purified CD34+ Cells by a Non-Integrating Plasmid.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1589-1589 ◽  
Author(s):  
Xiaosong Huang ◽  
Bin-Kuan Chou ◽  
Prashant Mali ◽  
Zhaohui Ye ◽  
Sarah N Dowey ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
B Cells ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Disease Modeling ◽  
Retroviral Vectors ◽  
Cd34 Cells ◽  
Episomal Vectors ◽  
Adult Peripheral Blood

Abstract Abstract 1589 Human induced pluripotent stem cells (iPSCs) that are functionally similar to embryonic stem cells (ESCs) hold great potential for cell and gene therapies, disease modeling and drug development. The earliest success was achieved by using adherent fibroblastic cells and retroviral vectors that transduce fibroblasts very efficiently. It is also highly desirable to reprogram postnatal blood cells, including those from cord blood (CB) and adult peripheral blood (PB), which are easily accessible and less exposed to environmental mutagens. In 2009, we and others have achieved the reprogramming of human postnatal blood cells using the 4 Yamanaka factors delivered by retroviral vectors. We also found that reprogramming efficiencies of CB and PB CD34+ cells are higher than age-matched fibroblasts or MSCs. This may result from an epigenetic profile of hematopoietic CD34+ cells that appears closer to iPSCs/ESCs than that of fibroblasts/MSCs to iPSCs/ESCs. To generate integration-free iPSCs that produce hematopoietic progeny efficiently, we attempted to reprogram adult PB as well as CB cells by OriP/EBNA1 episomal vectors, which were used previously to reprogram foreskin fibroblasts albeit at a low efficiency (Yu/Thomson, 2009). When one of the best combinations (#6, 3 plasmids) was used, 1–3 candidate iPSC clones per 1 million cells were obtained as reported (Yu/Thomson, 2009). We and others found that the efficiency of generating iPS clones was even lower with human adult somatic cells by the 3 vectors. To improve, we constructed a new episomal reprogramming vector system using 1–2 OriP/EBNA1 plasmids. One (pEB-C5) expresses 5 factors (OCT4/SOX2/KLF4/Myc/LIN28), and the second expresses SV40 T antigen (Tg). CB and adult PB CD34+ cells were first cultured for 4 days and expanded ≥4-folds. The expanded cells (1 million) were then transfected once by the 1–2 new OriP/EBNA1 plasmids we constructed. Fourteen days later, we obtained on average 250 and 9 TRA-1-60+, iPSC-like colonies from CB and adult PB cells, respectively, when both pCB-C5 and pEB-Tg were used. A single plasmid (pEB-C5) can also generate iPSCs although the efficiency is ∼4-folds lower. Five characterized iPSC lines derived from CB and adult PB CD34+ cells (with or without Tg) are karyotypically normal and pluripotent. After successful reprogramming and expansion, episomal DNA is gradually lost in proliferating iPSCs. After serial expansions for 11–12 passages, vector DNA was undetectable either as episomes or in the genome of the 5 iPSC lines. We next extended this approach to reprogram un-fractionated adult PB mononuclear cells (PBMCs) including those from a sickle cell patient (SCDB003). To achieve better cell proliferation that is critical to iPSC production, we used a culture condition that favors the formation and proliferation of erythroblasts from PBMCs. PBMCs purified by standard Ficoll gradient were cultured in a serum-free condition with cytokines SCF, EPO and IL-3. Although cell death was observed and cell number decreased significantly in the first 4 days, equal or more cells than input were obtained by day 8. The expanded cells morphologically resemble pro-erythroblast cells, and express high-level CD71. Less than 1.5% of them express markers of T cells (CD3, CD2, CD4 and CD8) and B cells (CD19 and CD20). When 2×106 expanded SCDB003 cells (achievable from PBMCs in 1 ml or less PB) were transfected by the 2 OriP/EBNA1 plasmids and reprogrammed in the presence of butyrate, we observed 8 colonies at day 14 that are TRA-1-60+ and iPSC-like. The second plasmid (pEB-Tg) was not essential although it enhanced the efficiency by ∼4 folds. We picked and characterized 3 iPSC-like colonies derived from PBMCs with or without Tg. All of them express pluripotency markers and behave as typical iPSCs. So far we do not have evidence if they are derived from committed T or B cells that somatic mutations altered and rearranged their genomes. We are currently examining karyotypes, in vivo pluripotency, and status of episomal vectors in 3 PBMC-derived iPSCs. As compared to recent studies using viruses that preferentially reprogram human T cells with a rearranged genome, our method of using 1–2 plasmids is virus-free and genomic alteration-free. The ability to obtain integration-free human iPSCs from a few ml PB by 1–2 plasmids will greatly accelerate uses of iPSCs in both research and future clinical applications, epically for blood disease modeling and treatment. Disclosures: No relevant conflicts of interest to declare.

Influence of G-CSF Mobilization and Liquid Storage Time on Post-Thaw Recovery of Lymphocyte Subpopulations and CD34+ Cells in Cryopreserved Apheresis Products

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4307-4307
Author(s):  
Christina Berens ◽  
Annkristin Heine ◽  
Jens Mueller ◽  
Johannes Oldenburg ◽  
Dominik Wolf ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Peripheral Blood ◽  
Storage Time ◽  
Statistical Significance ◽  
Lymphocyte Subpopulations ◽  
Hematopoietic Stem ◽  
Liquid Storage ◽  
Cd34 Cells ◽  
The Impact

Abstract Background: Peripheral blood stem cells (PBSC) and peripheral blood mononuclear cells (PBMC) for allogeneic transplantation of stem cells or donor lymphocyte infusion are frequently cryopreserved to allow cellular therapy at later time-points. Differences in the post-thaw recovery of CD34+ and CD3+ T cells in cryopreserved products have been hypothesized to depend on liquid storage time or, in the case of CD3+ T cells, on the mobilization by G-CSF. Methods: The recovery of CD3+ CD4+, CD3+ CD8+, CD19+, CD16+ CD56+, and CD34+ cells from 58 allogeneic apheresis products (among which 38 derived from G-CSF-mobilized donors) was analyzed by flow cytometry in order to evaluate the impact of the freezing/thawing process in the recovery of lymphocyte subpopulations as well as hematopoietic stem cells. In addition, cell viability was determined by measuring viable CD45+ (vCD45+) cells. All measurements were performed with aliquots that had been cryopreserved in parallel with the products using a controlled-rate freezer and 8% dimethyl sulfoxide and were stored in liquid nitrogen. Results: The post-thaw recovery was 78.6 ± 14.7% (mean±SD) for CD3+ CD4+ cells, 79.0 ± 12.9% for CD3+ CD8+ cells, 95.6 ± 15.8% for CD19+ cells, 84.6 ± 22.4% for CD16+ CD56+ cells, and 93.9 ± 24.6% for CD34+ cells. In G-CSF mobilized products, higher recovery rates were observed than in non-mobilized products, reaching statistical significance for CD3+ CD4+ T cells (82.8 ± 14.5 vs 72.1 ± 12.8%, p=0.008), CD19+ B cells (99.3 ± 15.9 vs 89.7 ± 14.1%, p=0.027), and CD16+ CD56+ NK cells (90.1% ± 19.8% vs 75.9 ± 22.4%, p=0.025). Within the lymphocyte subpopulations the post-thaw recovery was significantly lower for CD3+ CD4+ vs CD19+ (p=1.0x10-9 in G-CSF mobilized products, p=2.9x10-5 in non-mobilized products), and CD3+ CD8+ vs CD19+ (p=1.8x10-8 in G-CSF mobilized products, p=3.2x10-4 in non-mobilized products). With a ratio of vCD45+/CD45+ of 62.6 ± 18.2% in G-SCF mobilized products and 64.4 ± 18.3% in non-mobilized products no difference in the viability was observed. Spearman's analysis revealed only a weak negative correlation between liquid storage time (30 ± 14 h for G-CSF mobilized products, 22 ± 7 h for non-mobilized products) and viability (rs=-0.33, p=0.015), and between liquid storage time and CD34+ recovery (rs=-0.42, p=0.009). The post-thaw recovery of all other cell types did not decrease with longer liquid storage time. Conclusion: G-CSF mobilization and longer liquid storage time do not impair post-thaw recovery of lymphocyte subpopulations when compared to products from non-mobilized donors. However there is a slight decrease of viability with longer liquid storage time. Our results further suggest that T lymphocytes exhibit a higher sensitivity toward freezing and thawing than B lymphocytes, which may have clinical implications for cellular therapies using frozen products. Disclosures Oldenburg: SOBI: Consultancy.

Promoter IV of the class II transactivator gene is essential for positive selection of CD4+ T cells

Blood ◽  
2003 ◽  
Vol 101 (9) ◽  
pp. 3550-3559 ◽  
Author(s):  
Jean-Marc Waldburger ◽  
Simona Rossi ◽  
Georg A. Hollander ◽  
Hans-Reimer Rodewald ◽  
Walter Reith ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Epithelial Cells ◽  
Positive Selection ◽  
Negative Selection ◽  
Cd4 T Cell ◽  
Thymic Epithelial Cells ◽  
Mhcii Expression ◽  
Selection Of

Major histocompatibility complex class II (MHCII) expression is regulated by the transcriptional coactivator CIITA. Positive selection of CD4+ T cells is abrogated in mice lacking one of the promoters (pIV) of the Mhc2ta gene. This is entirely due to the absence of MHCII expression in thymic epithelia, as demonstrated by bone marrow transfer experiments between wild-type and pIV−/− mice. Medullary thymic epithelial cells (mTECs) are also MHCII− in pIV−/− mice. Bone marrow–derived, professional antigen-presenting cells (APCs) retain normal MHCII expression in pIV−/− mice, including those believed to mediate negative selection in the thymic medulla. Endogenous retroviruses thus retain their ability to sustain negative selection of the residual CD4+ thymocytes in pIV−/− mice. Interestingly, the passive acquisition of MHCII molecules by thymocytes is abrogated in pIV−/−mice. This identifies thymic epithelial cells as the source of this passive transfer. In peripheral lymphoid organs, the CD4+T-cell population of pIV−/− mice is quantitatively and qualitatively comparable to that of MHCII-deficient mice. It comprises a high proportion of CD1-restricted natural killer T cells, which results in a bias of the Vβ repertoire of the residual CD4+ T-cell population. We have also addressed the identity of the signal that sustains pIV expression in cortical epithelia. We found that the Jak/STAT pathways activated by the common γ chain (CD132) or common β chain (CDw131) cytokine receptors are not required for MHCII expression in thymic cortical epithelia.

scholarly journals Absence of H-2 restriction in primary and secondary mixed-lymphocyte reactions to strong M1s determinants.

1980 ◽  
Vol 151 (2) ◽  
pp. 407-417 ◽  
Author(s):  
K Molnar-Kimber ◽  
J Sprent
Keyword(s):  
T Cells ◽  
Positive Selection ◽  
Proliferative Response ◽  
Negative Selection ◽  
Selection Procedures ◽  
Per Se ◽  
Mixed Lymphocyte Reactions ◽  
Selection Of

Negative and positive selection procedures were used to establish whether the strong proliferative response of T cells to M1sa determinants is H-2 restricted. After negative selection of H-2 determinants in vivo, it was shown that T cells give high primary mixed lymphocyte reactions in vitro to M1sa determinants presented on H-2-incompatible stimulator cells. Other studies demonstrated that (a) negative selection of T cells to M1sa determinants on H-2-incompatible cells removed T cells with specificity for M1sa-bearing H-2-compatible cells, and (b) T cells primed in vitro or in vivo to M1sa determinants on H-2-compatible cells gave high secondary responses to M1sa determinants presented either on H-2-compatible or H-2-incompatible stimulator cells. From these data we conclude that T cells recognize M1sa determinants per se rather than an association of M1sa plus self or allo-H-2 determinants.

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