Clonal Tracking of the Geographic Distribution of Hematopoiesis in Nonhuman Primates Provides New Insights into HSPC Migration and Differentiation

Abstract Hematopoietic stem and progenitor cells (HSPC) primarily reside in bone marrow (BM) niches, but are also found in peripheral blood (PB) in small numbers. Large numbers of HSPC can be pharmacologically mobilized from the BM into the PB and home back to BM niches following transplantation. We used the rhesus macaque to study the process of hematopoiesis in both space and time following autologous transplantation, as a model with great relevance to humans. We labeled individual CD34+ HSPC and their progeny with genetic "barcodes" via lentiviral transduction with a very high diversity barcode library, allowing tracking of the output from individual HSPC clones in vivo following autologous transplantation, in a quantitative and sensitive manner (Wu et al., 2014). In this study we investigated the pattern of HSPC clonal output in various anatomic niches, sampling left (L) vs right (R) iliac crest BM, PB and lymph node (LN) over time post ablative autologous transplantation, for all major hematopoietic lineages and CD34+ HSPC. We tracked thousands of HSPC clones in 6 rhesus macaques from 3.5 months (m) to up to 18.5m post transplantation. L and R iliac crest BM and PB were serially collected and CD34+ HSPC along with CD3+CD4+ T, CD3+CD8+ T, CD20+ B, CD14+ monocytes (Mo), CD33+ granulocytes (Gr), and two NK cell subsets (CD3-CD20-CD14-CD16+/ or CD56+) were purified. In all animals, there was marked geographic segregation of CD34+ HSPC for at least 6m post-transplant, with individual clones localized only to the L or R side but not both (Pearson correlation at 3.5-6m: r=0.019±0.08 for CD34+ L vs R, n=6), despite rapid expansion of CD34+ HSPC during early engraftment. This result suggests that during this phase of recovery, HSPC spread contiguously in the BM, and there is little geographic "mixing" via egress into and re-entry from the PB. With time between 7m and 18.5m post-transplant, the distribution of CD34+ HSPC became more homogeneous, with clones detected on both L and R (r=0.52±0.22 at 12-18.5m, n=3). The geographic restriction of clones at earlier time points suggests that release and homing may be dependent on local contiguous niche occupancy status, with migration only occurring following hematopoietic recovery. We next examined the clonal distribution of the more mature lineage-committed cells in the L vs R BM and in PB. Gr, Mo and B cells were produced locally in BM, with the clonal pattern of each lineage matching the CD34+ cells collected from the same side BM time point through 6m (r>0.80 for each lineage vs same side CD34+ cells), and distinct from same lineages (r<0.16 for each lineage in L vs in R BM) and CD34+ cells on the other side (r<-0.01 for each lineage vs other side CD34+ cells). CD16+CD56-/dim NK clones were completely shared between PB and both L and R BM, suggesting they were not produced locally in BM, but instead in other sites with homogenous lodging or homing back to BM. Most surprisingly, we found population of CD3+CD8+ T cells that appeared to be produced locally, with barcodes matching the CD34+ HSPC at the same location, suggesting a novel T cell development pathway within the BM for this subset during early hematopoietic reconstitution. In contrast, CD3+CD4+ BM T cells had similar clonal constitution on the L vs R, and matched the PB, suggesting they had re-circulated back to the BM following maturation elsewhere, such as the thymus. T, B and NK cells from two LNs obtained simultaneously were also analyzed. Clonal contributions to T, B, and NK cells from L vs R LNs were highly correlated (r=0.95, r=0.88, and r=0.89 respectively). The clonal composition of T or B cells in LN were shared with circulating T or B cells (r=0.88, r=0.85 respectively), while LN NK cells (which are primarily CD56+/CD16-) shared barcodes with circulating CD16-CD56+ NK (r=0.81), not with PB CD16+CD56- cells(r=0.26), suggesting a non-precursor/progeny relationship. Our model for the first time documents the dynamics of HSPC geographic distribution and migration in primates following transplantation, findings with direct clinical relevance, and provides new insights into hematopoietic lineage development, including a potential novel T cell development pathway in the BM. Our findings may also help explain the extremely patchy distribution of hematopoiesis in humans following transplantation or in the setting of marrow failure or aging, and suggest that analysis of individual BM samples may not fully reflect ongoing global hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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A Detailed Phenotypic Analysis Using Six Color Flow Cytometry of Lymphocyte Subsets Mobilized with AMD3100 Compared to G-CSF.

Blood ◽

10.1182/blood.v104.11.408.408 ◽

2004 ◽

Vol 104 (11) ◽

pp. 408-408 ◽

Cited By ~ 1

Author(s):

Yoshiyuki Takahashi ◽

S. Chakrabarti ◽

R. Sriniivasan ◽

A. Lundqvist ◽

E.J. Read ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Lymphocyte Subsets ◽

Phenotypic Analysis ◽

Color Flow ◽

Cd34 Cells

Abstract AMD3100 (AMD) is a bicyclam compound that rapidly mobilizes hematopoietic progenitor cells into circulation by inhibiting stromal cell derived factor-1 binding to its cognate receptor CXCR4 present on CD34+ cells. Preliminary data in healthy donors and cancer patients show large numbers of CD34+ cells are mobilized following a single injection of AMD3100. To determine whether AMD3100 mobilized cells would be suitable for allografting, we performed a detailed phenotypic analysis using 6 color flow cytometry (CYAN Cytometer MLE) of lymphocyte subsets mobilized following the administration of AMD3100, given as a single 240mcg/kg injection either alone (n=4) or in combination with G-CSF (n=2: G-CSF 10 mcg/kg/day x 5: AMD3100 given on day 4). Baseline peripheral blood (PB) was obtained immediately prior to mobilization; in recipients who received both agents, blood was analyzed 4 days following G-CSF administration as well as 12 hours following administration of AMD3100 and a 5th dose of G-CSF. AMD3100 alone significantly increased from baseline the PB WBC count (2.8 fold), Absolute lymphocyte count (ALC: 2.5 fold), absolute monocyte count (AMC: 3.4 fold), and absolute neutrophil count (ANC: 2.8 fold). Subset analysis showed AMD3100 preferentially increased from baseline PB CD34+ progenitor counts (5.8 fold), followed by CD19+ B-cells (3.7 fold), CD14+ monocytes (3.4 fold), CD8+ T-cells (2.5 fold), CD4+ T-cells (1.8 fold), with a smaller increase in CD3−/CD16+ or CD56+ NK cell counts (1.6 fold). There was no change from baseline in the % of CD4+ or CD8+ T-cell expressing CD45RA, CD45RO, or CD56, CD57, CD27, CD71 or HLA-DR. In contrast, there was a decline compared to baseline in the mean percentage of CD3+/CD4+ T-cells expressing CD25 (5.5% vs 14.8%), CD62L (12.1% vs 41.1%), CCR7 (2.1% vs 10.5%) and CXCR4 (0.5% vs 40.9%) after AMD3100 administration; similar declines in expression of the same 4 surface markers were also observed in CD3+/CD8+ T-cells. A synergistic effect on the mobilization of CD34+ progenitors, CD19+ B cells, CD3+ T-cells and CD14+ monocytes occurred when AMD3100 was combined with G-CSF (Figure). In those receiving both AMD3100 and G-CSF, a fall in the % of T-cells expressing CCR7 and CXCR4 occurred 12 hours after the administration of AMD3100 compared to PB collected after 4 days of G-CSF; no other differences in the expression of a variety activation and/or adhesion molecules on T-cell subsets were observed. Whether differences in lymphocyte subsets mobilized with AMD3100 alone or in combination with G-CSF will impact immune reconstitution or other either immune sequela (i.e. GVHD, graft-vs-tumor) associated with allogeneic HCT is currently being assessed in an animal model of allogeneic transplantation.

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Co-Infusion of Allogeneic Cord Blood with Haploidentical CD34+ Cells Improved Transplant Outcome for Patients with Severe Aplastic Anemia Undergoing Cord Blood Transplantation

Blood ◽

10.1182/blood.v118.21.654.654 ◽

2011 ◽

Vol 118 (21) ◽

pp. 654-654 ◽

Cited By ~ 6

Author(s):

Nicole J. Gormley ◽

Jennifer Wilder ◽

Hahn Khuu ◽

Jeremy Pantin ◽

Theresa Donohue ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cord Blood ◽

Aplastic Anemia ◽

Severe Aplastic Anemia ◽

Transplant Outcome ◽

Neutrophil Recovery ◽

Post Transplant ◽

Cd34 Cells ◽

Matched Donor

Abstract Abstract 654 Unrelated cord blood (UCB) transplantation is a useful alternative for patients with hematological malignancies or non-malignant hematological disorders lacking an HLA matched donor. However, outcomes for patients with severe aplastic anemia (SAA) undergoing either a single or dual UCB transplant have been disappointing. A recent EBMT/ Eurocord study reported engraftment and 3 year survival rates of only 51% and 38% respectively (Perrault de Latour, Biol Blood Marrow Transplant 2011). We investigated whether co-infusion of a single UCB unit with CD34+ selected cells from a haploidentical relative following a highly immunosuppressive conditioning regimen could improve transplant outcome for patients with SAA refractory to immunosuppressive therapy that lack an HLA matched donor. Subjects with SAA and life-threatening neutropenia (ANC <500) refractory to 2 or more immunosuppressive agents were eligible for enrollment if they lacked an HLA matched related or unrelated donor. Conditioning consisted of cyclophosphamide (120 mg/kg), fludarabine (125 mg/m2), equine ATG (160 mg/kg) and one dose of 200 cGy of total body irradiation. Patients received a G-CSF mobilized, T-cell depleted CD34+ selected stem cell product prepared from a haplo-identical donor using the Miltenyi CliniMacs system combined with a single ≥ 4/6 HLA antigen matched UCB unit. Eight patients with treatment refractory SAA (median age 18 years; range 9–20), including 1 patient with SAA evolved to MDS, have been transplanted. All patients were platelet and RBC transfusion-dependent with severe neutropenia (median ANC 60 neutrophils/ul; range 0–260). Patients were at high risk for graft rejection, being heavily transfused, having failed a median 3 (range 2–4) immunosuppressive regimens, and 4 (50%) were HLA alloimmunized. Six patients received a single 4/6 HLA matched UCB unit and 2 received a 5/6 HLA-matched unit. Transplanted allografts contained a median 2.9 × 107 CB TNCs/kg (range 2.6–7.3), 1.5 × 105 CB CD34+cells /kg (range 0.4–3.2), and 3.3 ×106 CD34+cells/kg (range 2.6–3.6) from the haploidentical relative. All patients achieved the primary study endpoint of donor engraftment with an ANC of >500 by day 42, 7 of 8 achieving a UCB-derived ANC >500 cells/μl. The median time to neutrophil recovery was 10 days (range 10–18 days). One patient failed to engraft with the cord unit, but has had sustained engraftment from the haploidentical donor, and is transfusion independent with a normal neutrophil count >25 months post transplant. Acute GVHD grade II developed in 2 patients and one developed limited chronic GVHD. Early T-cell engraftment was predominantly UCB in 7 cases; on day 21, T-cell chimerism was a median 100% cord in origin (range 0–100%). In contrast, myeloid chimerism at engraftment was predominantly haplo-donor in origin and showed 3 phases of engraftment: 1) early myeloid engraftment from the haplo-CD34+ cell donor 2) delayed myeloid engraftment from the cord unit resulting in dual myeloid chimerism and 3) disappearance of the haplo-donor cells with transition towards full cord donor myeloid chimerism (see figure). Mixed lymphocyte reactivity assays performed on post transplant PBMCs showed increasing alloreactivity of cord blood T-cells against the haploidentical donor during the period when myeloid chimerism transitioned towards cord, indicating that the disappearance of haplo-donor myeloid cells occurred as a consequence of rejection by engrafting cord blood T-cells. At a median follow-up of 9 months (range 75 days to 3 years), 7 patients survive, and all are transfusion–independent. One patient died 14 months after transplantation from complications related to CMV pneumonitis. In conclusion, transplantation of haploidentical CD34+ cells can shorten the time to neutrophil recovery in SAA pts undergoing a single UCB transplant. Furthermore, durable full engraftment from donor haploidentical CD34+ cells can occur in the context of cord graft failure. These data suggest co-infusion of allogeneic cord blood with haploidentical CD34+ cells can improve the outcome of UCB transplantation for SAA. Disclosures: Wilder: NCI: Funded in part by NCI contract No. HHSN261200800001E.

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Rituximab Administration before Allogeneic HSCT Is Associated with Accelerated T Cell Reconstitution after Transplantation: Kinetic Evidence for a Graft-Versus-Leukemia Effect

Blood ◽

10.1182/blood.v124.21.3905.3905 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3905-3905

Author(s):

Sakura Hosoba ◽

Christopher R. Flowers ◽

Catherine J Wu ◽

Jens R. Wrammert ◽

Edmund K. Waller

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Cd8 T Cells ◽

Cell Subset ◽

Post Transplant ◽

T Cell Reconstitution ◽

Relative Value ◽

History Of

Abstract Introduction: Rituximab (R) administration results in depletion of blood B cells and suppression of B cell reconstitution for several months after, with suggestions that T cell reconstitution may also be impaired. We hypothesized that pre-transplant R would be associated with delayed B and T cell reconstitution after allo-HSCT compared with non-R-treated allo-HSCT recipients. Methods: We conducted a retrospective analysis of 360 patients who underwent allo-HSCT using BM or G-CSF mobilized PB. Recipients of cord blood, T cell depleted grafts and 2nd allo-HSCT were excluded. Analysis of lymphocyte subsets in at least one blood at 1, 3, 6, 12, and 24 months post-allo-HSCT was available for 255 eligible patients. Data on lymphocyte recovery was censored after DLI or post-transplant R therapy. Post-HSCT lymphocyte recovery in 217 patients who never received R (no-R) was compared to 38 patients who had received R before allo-HSCT (+R) including 12 CLL, 19 NHL, and 7 B-cell ALL patients. +R patients received a median of 9 doses of R with the last dose of R at a median of 45 days pre-transplant. Results: Mean lymphocyte numbers in the blood at 1, 3, 6, 12, and 24 months were B-cells: 55 ± 465/µL, 82 ± 159/µL, 150 ± 243/µL, 255 ± 345/µL, and 384 ± 369/µL (normal range 79-835); and T-cells: 65 ± 987/µL, 831 ± 667/µL, 1058 ± 788/µL, 1291 ± 985/µL, and 1477 ± 1222/µL (normal range 675-3085). Lymphocyte reconstitution kinetics did not vary significantly based upon the intensity of the conditioning regimen or related vs. unrelated donors allowing aggregation of patients in the +R and no-R groups (Figure). B cell reconstitution in the +R patients was higher at 1 month post-allo-HSCT (relative value of 143% p=0.008) and lower at 3 months post-transplant (19.2%, p=0.069) compared to no-R patients. Blood B cells in the +R group rebounded by the 6th month post-allo-HSCT and remained higher than the no-R group through the 24th month post-HSCT (197% at the 6th month, p=0.037). Higher levels of B-cells at 1 month in the +R group was due to higher blood B-cells at 1 month post-HSCT among 12 CLL patients compared with no-R patients (423%, p<0.001; Figure), while B-cell counts in the remaining +R patients (B-cell NHL and B-cell ALL) were lower than the no-R patients at both 1 and 3 months. Reconstitution of CD4+ and CD8+ T cells among +R patients were similar to no-R patients in the first month post-allo-HSCT and then rebounded to higher levels than the no-R group of patients (relative value 194%, p=0.077 at the 24th month for CD4+ T cell subset, and 224%, p=0.020 for CD8+ T cell subset; Figure). CLL patients had a striking increase in blood levels of donor-derived CD4+ and CD8+ T cells at 3 months post-transplant concomitant with the disappearance of blood B cells compared with no-R patients (relative value of 178% and 372%, p=0.018 and p=0.003, respectively; Figure). Long term T cell reconstitution remained higher for +R patients compared with no-R patients, even when CLL patients were excluded (relative value of 203%, p=0.005 at 24 months post-HSCT; Figure). Conclusions: We observed higher levels of blood B cells and T cells ³ 6 months post-allo-HSCT in +R patients compared with no-R patients. B cell recovery at 6 months post-transplant is consistent with clearance of residual plasma R given the 1-2 months half-life of R, and the median of 1.5 months between the last dose of R and allo-HSCT. The increased blood CD8+ T cells in the blood of CLL patients at 3 months post-allo-HSCT associated with clearance of the B-cells seen 1 month post-HSCT is consistent with a donor T cell-mediated GVL effect. Pre-transplant R therapy does not appear to have any long-term deleterious effect on immune reconstitution, indicating that post-allo-HSCT vaccination at ≥6 months may be efficacious. Figure: Kinetics of lymphocyte reconstitution after allo-HSCT varied by history of pre-transplant R administration and primary disease. Panels show mean counts of each lymphocyte subset at 1, 3, 6, 12 and 24 months post-allo-HSCT for: (1) B cell, (2) T cell, (3) CD4+ and (4) CD8+ T cells. Solid lines with triangle show no-R group; dashed lines with circles shows subgroups of CLL and NHL/ALL +R patients. Asterisks show p values from t-test of the comparison between CLL +R or the NHL/ALL +R patients with no-R patients. *p<0.05; ** p<0.01; *** p<0.001. Figure:. Kinetics of lymphocyte reconstitution after allo-HSCT varied by history of pre-transplant R administration and primary disease. Panels show mean counts of each lymphocyte subset at 1, 3, 6, 12 and 24 months post-allo-HSCT for: (1) B cell, (2) T cell, (3) CD4+ and (4) CD8+ T cells. Solid lines with triangle show no-R group; dashed lines with circles shows subgroups of CLL and NHL/ALL +R patients. Asterisks show p values from t-test of the comparison between CLL +R or the NHL/ALL +R patients with no-R patients. *p<0.05; ** p<0.01; *** p<0.001. Disclosures No relevant conflicts of interest to declare.

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Human Lymphoid and Myeloid Cell Development in NOD/LtSz-scid IL2rγnull Mice Engrafted with Mobilized Human Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v104.11.248.248 ◽

2004 ◽

Vol 104 (11) ◽

pp. 248-248 ◽

Cited By ~ 6

Author(s):

Leonard Shultz ◽

Bonnie L. Lyons ◽

Lisa M. Burzenski ◽

Bruce Gott ◽

X. Chen ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

T Cell ◽

B Cells ◽

Nk Cells ◽

De Novo ◽

Cell Development ◽

In Vivo Model ◽

Hematopoietic Stem ◽

Human T Cell

Abstract We have developed, characterized, and validated a new genetic stock of IL-2r common γ (gamma) chain deficient NOD/LtSz-scid (NOD-scid IL2rγnull) mice that support high levels of human hematopoietic stem cell (HSC) engraftment and multilineage differentiation. Histology, flow cytometry, and functional assays document a severe depletion of lymphocytes and NK cells in NOD-scid IL2rγnull mice. These mice survive beyond 16 months of age and untreated as well as sub-lethally irradiated NOD-scid IL2rγnull mice are resistant to the development of lymphomas and are “non-leaky” throughout life. Intravenous injection of sub-lethally irradiated NOD-scid IL2rγnull mice with 7 x 105 human mobilized CD34+ stem cells leads to high levels of multilineage engraftment. At 10 weeks after engraftment, percentages of human hematopoietic CD45+ cells are six-fold higher in the bone marrow of NOD-scid IL2rγnull mice as compared to NOD-scid controls. Human CD45+ cells include immature and mature B cells, NK cells, myeloid cells, plasmacytoid dendritic cells and HSCs. Spleens from engrafted NOD-scid IL2rγnull mice contain high percentages of immature and mature B cells but low percentages of T cells. Treatment with human Fc-IL7 fusion protein leads to a high percentage of human CD4+CD8+ immature thymocytes and high percentages of CD4+CD8− and CD4−CD8+ mature human T cells in the spleen and blood. Validation of de novo human T cell development was carried out by quantifying T cell receptor excision circles in thymocytes and by analyses of TCRβ repertoire diversity. Human T cell function was evidenced by proliferative responses to PHA and streptococcal superantigen. NOD-scid IL2rγnull mice engrafted with human HSC generate differentiated functional human T and B cells and provide an in vivo model of multilineage human hematopoietic cell engraftment.

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Impact of Acute GVHD on Immune Reconstitution after Cord Blood Transplantation in Adult.

Blood ◽

10.1182/blood.v104.11.984.984 ◽

2004 ◽

Vol 104 (11) ◽

pp. 984-984 ◽

Cited By ~ 2

Author(s):

Tokiko Nagamura-Inoue ◽

Satoshi Takahashi ◽

Jun Ooi ◽

Akira Tomonari ◽

Toru Iseki ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Cord Blood ◽

Nk Cells ◽

Immune Reconstitution ◽

Cord Blood Transplantation ◽

Adult Patients ◽

Blood Transplantation ◽

Cd34 Cells ◽

Grade Ii

Abstract BACKGROUND: Immune reconstitution following unrelated cord blood transplantation (UCBT) in adult patients is of great concern because of immaturity of cord blood immunological cells. STUDY DESIGN AND METHODS: Twenty-six adult patients (15 to 58 year-old) with hematological malignancies, who underwent UCBT and sustained engraftment were enrolled in this study. Infused number of immunological cells in thawed CB units including T cells (CD3+), B cells (CD19+), NK cells (CD3-CD56+), monocytes (CD14+) and also CD34 + cells was analysed using bead-contained TRUCOUNT tube (BD, CA). Dead cells after thawing were excluded by gating out with 7AAD dye. Immune reconstitution was analysed every 30 days by 120 days after CBT. Four-colour FACS Caliber and TRUCOUNT tube were utilized to calculate the absolute number of immune cells concentration in blood after UCBT. We put strict volume of 50μl fresh unmanipulated blood in each TRUCOUNT tube. RESULTS: Thawed-transplanted NC 2.3±x107/kg, CD34 was 0.72±0.3x105/kg (4.1x106 total), T cells; 3.1±1.6x106/kg with CD4/8 ratio of 3.2±2.0, B cells; 1.2±0.5x106/kg, NK cells; 1.0±0.5x106/kg and monocytes; 1.6±0.6x106/kg. There were no correlations between infused CD34+ cells number and T, B, NK and monocytes numbers. Monocytes increased in blood rapidly after CBT at 30 days, then, declined to the normal value. NK cells was recovered in the early after CBT and then did not so change in number from 30 to 120 days after CBT, while T cells increased time dependent manner, and B cells appeared late but influenced by acute GVHD grade. Within 120 days after CBT, T cells showed also CD4+dominant in most cases with relatively high CD25+CD4+ regulatory T (rT) cells compared to normal control. The patients with grade II to IV aGVHD showed significantly higher number of rT cells on 30 days (P<0.05) compared to those with grade 0–I aGVHD. On day 30, the number of rT cells showed 7.7±5.9/μl in grade 0–I aGVHD and 19.4±13.3/ μl in grade II–IV. The patients with grade II to IV aGVHD showed significant delayed recovery of B cells on 90 days after CBT compared to those with 0–I aGVHD (P<0.001). CONCLUSION: aGVHD in adult patients may influence on the number of regulatory T cells in the early period after UCBT and delayed recovery of B cells.

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Notch Activation Converts B Cells into a T Cell Fate at the Earliest Stages of B Cell Committed Progenitors.

Blood ◽

10.1182/blood.v106.11.3151.3151 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3151-3151

Author(s):

Jalal Taneera ◽

Emma Smith ◽

Mikael Sigvardsson ◽

Emil Hansson ◽

Urban Lindahl ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Cell Fate ◽

T Cell Development ◽

Cell Development ◽

Cell Stage ◽

Cell Commitment ◽

Notch Activation

Abstract Notch activation has been suggested to promote T cell development at the expense of B cell commitment at the level of a common lymphoid progenitor prior to B cell commitment. Here, we explored the possibility that Notch activation might be able to switch the fate of already committed B cell progenitors towards T cell development upon Notch activation. To address this we overexpressed constitutively activated Notch-3 (N3IC) in B cell progenitors purified from transgenic mice in which human CD25 is expressed under control of the λ5 promoter. Strikingly, whereas untransduced and control transduced B220+λ5+CD3− B cell progenitors gave rise exclusively to B cells, CD4+ and CD8+ T cells but no B cells were derived from N3IC-transduced cells when transplanted into sublethally irradiated NOD-SCID mice. Gene expression profiling demonstrated that untransduced B220+ λ5+CD3− B cell progenitors expressed λ5 and CD19 but not the T cell specific genes GATA-3, lck and pTα, whereas CD3+ T cells derived from N3IC-transduced B220+λ5+CD3−cells failed to express λ5 and CD19, but were positive for GATA-3, lck and pTα expression as well as a and b T cell rearrangement. Furthermore, DJ rearrangements were detected at very low levels in CD3+ cells isolated from normal non-transduced BM, but were more abundant in the N3IC-transduced CD3+ BM cells. Noteworthy, N3IC-transduced B220+λ5+CD3−CD19+ proB cell progenitors failed to generate B as well as T cells, whereas N3IC-transduced B220+λ5+CD3−CD19− pre-proB cells produced exclusively T cells, even when evaluated at low cell numbers. In conclusion Notch activation can switch committed B cell progenitors from a B cell to a T cell fate, but this plasticity is lost at the Pro-B cell stage, upon upregulation of CD19 expression.

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Expression of CD10 and CD7 Defines Distinct In Vivo Lymphoid Reconstituting Capacity within Human Cord Blood CD34+CD38-Cells.

Blood ◽

10.1182/blood.v108.11.3184.3184 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3184-3184

Author(s):

Shuro Yoshida ◽

Fumihiko Ishikawa ◽

Leonard D. Shultz ◽

Noriyuki Saito ◽

Mitsuhiro Fukata ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

B Cells ◽

Cord Blood ◽

Nk Cells ◽

Progenitor Cells ◽

Human Cord Blood ◽

Cd34 Cells

Abstract Human cord blood (CB) CD34+ cells are known to contain both long-term hematopoietic stem cells (LT-HSCs) and lineage-restricted progenitor cells. In the past, in vitro studies suggested that CD10, CD7 or CD127 (IL7Ra) could be candidate surface markers that could enrich lymphoid-restricted progenitor cells in human CB CD34+ cells (Galy A, 1995, Immunity; Hao QL, 2001, Blood; Haddad R, 2004, Blood). However, in vivo repopulating capacity of these lymphoid progenitors has not been identified due to the lack of optimal xenogeneic transplantation system supporting development of human T cells in mice. We aim to identify progenitor activity of human CB CD34+ cells expressing CD10/CD7 by using newborn NOD-scid/IL2rgKO transplant assay that can fully support the development of human B, T, and NK cells in vivo (Ishikawa F, 2005, Blood). Although LT-HSCs exist exclusively in Lin-CD34+CD38- cells, not in Lin-CD34+CD38+ cells, CD10 and CD7 expressing cells are present in Lin-CD34+CD38- cells as well as in Lin-CD34+CD38+ cells (CD10+CD7+ cells, CD10+CD7- cells, CD10-CD7+ cells, CD10-CD7- cells accounted for 4.7+/−2.7%, 10.5+/−1.9%, 7.6+/−4.4%, and 77.1+/−5.2% in Lin-CD34+CD38- CB cells, respectively). We transplanted 500–6000 purified cells from each fraction into newborn NOD-scid/IL2rgKO mice, and analyzed the differentiative capacity. CD34+CD38-CD10-CD7- cells engrafted long-term (4–6 months) in recipient mice efficiently (%hCD45+ cells in PB: 30–70%, n=5), and gave rise to all types of human lymphoid and myeloid progeny that included granulocytes, platelets, erythroid cells, B cells, T cells, and NK cells. Successful secondary reconstitution by human CD34+ cells recovered from primary recipient bone marrow suggested that self-renewing HSCs are highly enriched in CD34+CD38–CD10–CD7- cells. CD10–CD7+ cells were present more frequently in CD34+CD38+ cells rather than in CD34+CD38- cells. Transplantation of more than 5000 CD34+CD38+CD10–CD7+ cells, however, resulted in less than 0.5% human cell engraftment in the recipients. Within CD34+CD38–CD10+ cells, the expression of CD7 clearly distinguished the distinct progenitor capacity. At 8 weeks post-transplantation, more than 70% of total human CD45+ cells were T cells in the CD10+CD7+ recipients, whereas less than 30% of engrafted human CD45+ cells were T cells in the CD10+CD7– recipients. In the CD10+CD7- recipients, instead, more CD19+ B cells and HLA–DR+CD33+ cells were present in the peripheral blood, the bone marrow and the spleen. Both CD34+CD38–CD10+CD7+ and CD34+CD38–CD10+CD7- cells highly repopulate recipient thymus, suggesting that these progenitors are possible thymic immigrants. Taken together, human stem and progenitor activity can be distinguished by the expressions of CD7 and CD10 within Lin-CD34+CD38- human CB cells. Xenotransplant model using NOD-scid/IL2rgKO newborns enable us to clarify the heterogeneity of Lin-CD34+CD38- cells in CB by analyzing the in vivo lymphoid reconstitution capacity.

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Notch Activation in Cord Blood Progenitors Induces a CD7+ Common NK/T Precursor Capable of NK Cell Commitment without Full Maturation and Differentiation of Lineage Committed Pre-T Lymphocytes with a Th1 Phenotype.

Blood ◽

10.1182/blood.v108.11.644.644 ◽

2006 ◽

Vol 108 (11) ◽

pp. 644-644

Author(s):

Veronika Bachanova ◽

Valarie McCullar ◽

Rosanna Wangen ◽

Jeffrey S. Miller

Keyword(s):

T Cells ◽

T Cell ◽

Cord Blood ◽

Nk Cells ◽

Nk Cell ◽

T Cell Development ◽

Notch Pathway ◽

Cell Development ◽

Dominant Negative ◽

Cell Commitment

Abstract Activation of Notch signaling regulates differentiation and homeostasis of hematopoetic stem cells. After stimulation, intracellular Notch is proteolytically released and by binding the CSL complex and co-activator MAML, and initiates transcription of downstream genes. We hypothesize that Notch is important for distinct stages of lymphoid development. Human cord blood CD34+ progenitor cells were transduced with retrovirus based eGFP-control, eGFP-Notch and Notch Dominant Negative/MAML (eGFP-DN) constructs. CD34+/eGFP+ were sorted and then co-cultured with the mouse embryonic liver cell line EL08.1D2 and exogenous human cytokines (IL-3. IL-7, IL-15, Flt3 ligand and c-kit ligand). As early as 48 hours after transduction, CD34+/Notch+ cells gave rise to population of lymphoid precursors CD34+CD7+CD10- (42±5% of all cells) while essentially no cells with this phenotype were detected with the control or DN construct. Proliferation of eGFP-Notch transduced cells in a 6-day thymidine incorporation assay was higher compared to eGFP-DN transduced cells (8410±839 vs. 1103±209 cpm; n=3; p=0.00005). Within 7 days 11±1.5% NK emerged from CD34+/Notch+cells compared to 0.8±0.2% of CD34+/eGFP+ control cells (n=5, p=0.0001). NK cell generation peaked at day 28 with a significantly higher expression of CD7 on NK cells (Notch: 75±5% vs. eGFP: 4.5±1%, n=5, p=0.00004), and no B lymphocytes were seen. Analysis of Notch induced NK cells demonstrated early expression of L-selectin and increased expression of CD45RA on all lymphoid progenitors. At 4 weeks, functional testing revealed reduced cytotoxicity against K562 (Notch: 37±0.5% vs. eGFP: 63.5±1.3%; n=7, p=0.007) suggesting immature function. CD34+/Notch+ derived NK lymphocytes also showed diminished acquisition of the lectin-type receptor NKG2A (Notch: 8.3±3% vs. eGFP: 27.4±4.5%; p=0.04) and killer immunoglobulin receptors (Notch: 2.2±0.5% vs. eGFP: 10.8±4: p=0.05). We next asked whether the Notch induced CD7+ precursor was NK restricted or a common NK/T cell precursor. After 5 weeks in culture, a distinct population of CD3+ T-cells emerged (Notch: 18±5% vs. eGFP: 1.6±0.2; n=5, p<0.001%) which were CD4 and CD8 negative and did not express surface TCR a/b or g/d, but expressed high levels pre-T-alpha mRNA. These Notch activated cells were bona fide T-cells based on their capacity to produce IL-2 after PMA/Ca/I stimulation (62.4±4% by intracellular staining) while essentially no IL-2 production occurred from eGFP control cells (1.6±0.2%; p<0.0001). T-cell development was dependent on both Notch and the EL08.1D2 as no T-cells resulted from CD34+/eGFP-Notch in the absence of stroma. These findings suggest that in addition to Notch and exogenous cytokines, other soluble factors are required for T cell development. In conclusion, our data showed that activated Notch pathway leads to differentiation of a common CD7+ lymphoid precursor capable of both early NK cell and T-cell differentiation. This suggests that differences in Notch ligands in local microenvironments (marrow, thymus, lymph node) may be an important mechanism to orchestrate NK and T cell development.

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In Vitro Assessment of Tolerance and Rejection Occurring After Co-Transplantation of Allogeneic Umbilical Cord Blood and Haploidentical CD34+ Cells as Treatment for Severe Aplastic Anemia

Blood ◽

10.1182/blood.v116.21.2352.2352 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2352-2352

Author(s):

Nicole J. Gormley ◽

Aleah Smith ◽

Maria Berg ◽

Lisa Cook ◽

Catalina Ramos ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cord Blood ◽

Cord Blood Transplantation ◽

Neutrophil Recovery ◽

Post Transplant ◽

Blood Transplantation ◽

Cd34 Cells ◽

Cord Blood Cells

Abstract Abstract 2352 Introduction/Methods: The administration of highly purified haploidentical peripheral blood CD34+ cells combined with an unrelated cord blood transplant results in earlier neutrophil engraftment than is typically seen with a cord blood transplant alone. Chimerism data from pilot trials evaluating this strategy have reported 3 phases of engraftment: 1) early myeloid engraftment from transplanted haplo-CD34+ cells followed by 2) cord blood engraftment resulting in dual chimerism and 3) the subsequent disappearance of haploidentical donor cells with resultant full donor cord chimerism. The mechanism accounting for the disappearance of haploidentical cells has not been defined. Here the clinical results and an in vitro assessment of alloreactivity in three patients that underwent combined haploidentical CD34+ cell and cord blood transplantation for severe aplastic anemia (SAA) are described. The conditioning regimen consisted of cyclophosphamide (60mg/kg/day on days -7 and -6), fludarabine (25mg/m2/day on days -5 to -1), horse ATG (40mg/kg/day on days -5 to -2), and total body irradiation (200cGy on day -1). GVHD prophylaxis consisted of tacrolimus and mycophenolate mofetil. PCR of STRs was used to assess chimerism in T-cell and myeloid lineages and mixed lymphocyte reaction assays(MLR) were performed on peripheral blood samples collected at different time-points post-transplant to assess for alloreactivity against the recipient, the haploidentical donor, or the cord unit. Stimulator cord blood cells for the MLR were obtained from residual cord blood cells remaining in the infusion bag after patient administration and expanded in vitro using anti-CD28/CD3 Dynabeads. Results: Prior to transplantation, all three pts had transfusion dependent SAA associated with severe neutropenia that was refractory to conventional immunosuppressive therapy. Pt 1 had an early transient myeloid recovery (ANC 400 on day+11) from the haploidentical donor followed by engraftment of the cord unit (Cord ANC > 500) on day 21. The patient is currently 2 years post transplant and has 100% cord blood chimerism and is transfusion independent. An MLR assay performed when donor T-cell chimerism was 100% cord, showed evidence for rejection of the haploid cells by cord blood T-cells, with the MLR response to haploidentical donor cells being seven fold higher than the response to fully HLA-mismatched 3rd party cells. In pt 2, neutrophil recovery from the transplanted haploidentical donor occurred on day +10, with chimerism studies showing no evidence for cord engraftment in either myeloid or T-cell lineages at any point post-transplant. The patient is currently 15 months post transplant and is transfusion independent with normal blood counts and sustained “split” chimerism (T-cells recipient in origin with myeloid cells being 100% haploidentical donor). MLR assays showed that the recipient was tolerant to the haploid donor, with no statistically significant difference in the alloreactive response to the haploid donor compared to self. In pt 3, neutrophil recovery from the transplanted haploidentical donor occurred on day +10, with chimerism studies showing split chimerism (T-cell chimerism >90% cord and myeloid chimerism 88–100% haploid donor in origin). MLR assays again showed evidence of rejection of the haploid cells by cord blood T-cells, with a trend towards greater alloreactivity against the haploid donor compared to an HLA mismatched 3rd party on post-transplant day +63. Conclusions: Combined haploidentical CD34+ cell and unrelated cord blood transplantation following highly immunosuppressive conditioning represents a viable treatment option for patients with SAA who lack an HLA-matched donor. Using this approach, 2 of 3 pts had cord blood engraftment associated with early neutrophil recovery from the haploidentical donor. In one pt, the cord unit failed to engraft. Remarkably, sustained engraftment from the haploidentical donor in this pt resulted in transfusion independence. MLR appears to be a useful approach to assess the in vitro alloreactivity of this unique stem cell graft source. In the two pts who had cord engraftment, in vitro MLR assessments established that the disappearance of haploid cells occurred as a consequence of rejection of the haploidentical cells by engrafting cord blood T-cells, rather than from non-immunological haploidentical cell graft failure. Disclosures: No relevant conflicts of interest to declare.

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Preclinical Study for the Use of Abatacept to Prevent Rejection of Allogeneic CD34+ Cells in a Xenograft Model

Blood ◽

10.1182/blood.v126.23.4271.4271 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4271-4271

Author(s):

Annie L. Oh ◽

Dolores Mahmud ◽

Vitalyi Senyuk ◽

Elisa Bonetti ◽

Nadim Mahmud ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

B Cells ◽

Xenograft Model ◽

Costimulatory Blockade ◽

Nsg Mice ◽

Human Cd34 ◽

Cd34 Cells

Abstract The aims of this study were to analyze the role of T cells on the engraftment of allogeneic CD34+ cells after transplantation in immunodeficient nonobese diabetic/ltsz-scid/scid (NOD/SCID) IL2 receptor gamma chain knockout (NSG) mice and to test the in-vivo ability of abatacept (CTLA4-Ig) in preventing graft failure. Human CD34+ cells (0.2x106 cells/animal) were co- transplanted with allogeneic CD3+ T cells into sublethally irradiated NSG mice at ratios ranging from 1:50 to 1:0.5, or without T cells as a control. The engraftment of huCD45+ cell subsets in the bone marrow and spleen was measured by flow cytometry after 4-8 weeks. An expansion of T cells without engraftment of CD34+ cells was detected in each group of mice transplanted with CD34:T cells at ratios ≥ 1:0.5. To test whether T cells prevented any engraftment of CD34+ cells, or caused rejection after initial CD34+ cell homing in the bone marrow, kinetics experiments were performed by analyzing the marrow and spleen of mice at 1,2 or 4 weeks after transplant of CD34+ and T cells at 1:1 ratio. These experiments showed that at two weeks after transplant, CD34+ cells had repopulated the bone marrow but not the spleen, while T cells were found primarily in the spleen. Instead, in mice sacrificed at 4 weeks after transplant the marrow and the spleen contained only T cells and the CD34+ cells had been rejected. Based on our previous in-vitro studies showing that CD34+ cell immunogenicity is mainly dependent on B7:CD28 costimulatory signaling, we then tested whether costimulatory blockade with abatacept (CTLA4-Ig, Bristol Myers Squibb) would block stem cell rejection. Three groups of mice were transplanted with CD34+ and allo-T cells at 1:1 ratio and injected with Abatacept at 250 ug i.p. every other day from: a) day -1 to +28, b) day -1 to day +14 or c) day +14 to +28, then the animals were sacrificed at day +56 (8 weeks) after transplant to assess the engraftment. In Group a) the overall engraftment of huCD45+ cells was only <10%, but Abatacept completely prevented T cell-mediated stem cell rejection with >98% huCD45+ cells of myeloid or B cell lineage and <1% T cells in the marrow and spleen. In Group b) 70% of huCD45+ cells both in the marrow and spleen were T cells, and the remaining fraction of myeloid or B cells were derived from CD34+ cells. In Group c), instead, 100% of huCD45+ cells were T cells, with complete rejection of CD34+ cells. T cells recovered from the spleen of mice in groups b) and c) were also tested as responders in MLC stimulated with the original CD34+ cells and showed a brisk proliferation, consistent with lack of tolerance. Finally, another group of mice that received Abatacept from day -1 to + 28 was rechallenged with a boost of CD34+ cells on day +28 to test whether the low CD34+ cell engraftment was secondary to a latent rejection or partial stem cell exhaustion. The CD34+ cell boost resulted in a full hematopoietic recovery with 37% huCD45+CD3- cells, including myeloid and B cells, as well as CD34+ cells in the bone marrow and spleen. In this preclinical xenograft model we demonstrated that costimulatory blockade with Abatacept at the time of allogeneic transplant of human CD34+ cells can prevent T cell mediated rejection provide the basis for the future non-myeloablative protocols for incompatible stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

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