TRANSPLANTATION AND CELLULAR ENGINEERING CME
Dual roles of autologous CD81 T cells in hematopoietic progenitor cell mobilization and engraftment Athena Russell,1 Sunita Malik,1 Mark Litzow,2 Dennis Gastineau,2 Vivek Roy,3 and Abba C. Zubair1
BACKGROUND: Poor marrow cellularity alone cannot explain poor hematopoietic progenitor cell (HPC) mobilization. This study assessed the role of CD81 T cells in HPC cell mobilization and engraftment. STUDY DESIGN AND METHODS: Mobilization and engraftment were assessed in 192 autologous HPC donors. CD341, CD41, and CD81 T-cell contents in apheresis products were evaluated. Using a chemotaxis assay, we assessed the effect of purified autologous CD81 T cells from low and high mobilizers on HPC migration from high to low stromal cell–derived factor (SDF-1a) concentration gradients. We also assessed CD81 T-cell content association with days to neutrophil engraftment. RESULTS: The median number of CD341 cells/kg was 4.7 3 106. Patients were categorized according to their total CD341 cell collection quartile distribution into low, moderate, and high mobilizers. We found that HPC products from low mobilizers contained more CD81 T cells than HPC products from moderate and high mobilizers. Chemotaxis assays showed depletion of CD81 T cells enhances HPC mobilization independent of SDF-1a concentration. Neutrophil engraftment analysis showed that the higher the CD81 T-cell content per unit CD341 cell, the faster the rate of engraftment. CONCLUSION: Our findings suggest CD81 T cells inhibit HPC mobilization and facilitate homing and engraftment.
F
ailure to collect adequate CD341 cell numbers following poor hematopoietic progenitor cell (HPC) mobilization is generally thought to be due to marrow damage after previous chemoand radiation therapies. Alkylating agents and high radiation dose are known to cause greater marrow damage.1 However, marrow damage alone cannot account for all poor mobilizations because there are donors who have high marrow cellularity for their age but fail to adequately mobilize enough CD341cells to achieve target cell dose. In addition, the relationship between CD341 cell dose and engraftment rate is not linear, suggesting that other factors may play a role during engraftment. Previous animal studies suggest CD81 T cells are important for CD341 cell homing to the marrow.2-5 We hypothesize that CD81 T cells have dual roles by inhibiting CD341 cell mobilization and facilitating engraftment. The stem cell compartment is composed of different types of cells whose presence is critical to stem cell physiology. These cells include mature differentiated hematopoietic cells such as T cells, dendritic cells, macrophages,
ABBREVIATIONS: qPCR 5 quantitative reverse transcriptase– mediated real-time polymerase chain reaction; SDF 5 stromal cell–derived factor; TNC 5 total nucleated cell; TNE 5 days to neutrophil engraftmen. From the 1Transfusion Medicine, Department of Pathology; and the 3Division of Hematology/Oncology, Mayo Clinic, Jacksonville, Florida; and the 2Division of Hematology/ Oncology, Mayo Clinic, Rochester, Minnesota Supported by NIH Grant CA102824. Address reprint requests to: Abba Zubair, MD, PhD, Transfusion Medicine, Department of Pathology, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL 32224; e-mail: zubair.abba@ mayo.edu. Received
for
publication
August
31,
2014;
revision
received November 20, 2014; and accepted November 28, 2014. doi:10.1111/trf.13073 C 2015 AABB V
TRANSFUSION 2015;55;1758–1765 1758 TRANSFUSION Volume 55, July 2015
ROLE OF CD81 T CELLS IN HPC MOBILIZATION AND ENGRAFTMENT
TABLE 1. Patient characteristics Mobilization CD341 cells (3106/kg) Number (192) Median age (years) Sex, female (%) Race* White Hispanic Black Other Disease categories* Leukemia Lymphoma Plasma cell disease
Low (3.7)
Moderate (3.8-5.5)
High (5.6)
44 59.5 56.8
99 59 37.4
49 55 40.8
37 2 4 1
(84) (5) (9) (2)
1 (2) 24 (55) 19 (43)
91 2 3 3
(92) (2) (3) (3)
47 (96) 0 1 (2) 1 (2)
4 (4) 22 (22) 73 (74)
0 15 (31) 34 (69)
* Data are reported as number (%).
and others. It has long been determined that stem cells require accessory cells to maintain their physiologic state.6 The role of T cells as facilitators of HPC engraftment became apparent when marrow grafts were depleted of T cells to minimize graft-versus-host disease.7 It was observed that T-cell depletion in autologous and allogeneic transplants resulted in delayed engraftment during the first 6 months after transplantation. This was further supported by clinical trials involving allogeneic transplant, which showed that the presence of CD81 T cells but not CD41 cells resulted in faster engraftment.5 Further studies in mice have also demonstrated that CD81 T cells facilitate HPC engraftment.8 The mechanism of action by which CD81 T cells facilitate allogeneic engraftment was largely attributed to elimination of residual host immune cells.5 However, it was unclear how CD81 T cells facilitated engraftment in autologous transplant settings. Granulocyte–colony-stimulating factor (G-CSF) is the principal growth factor used clinically to mobilize HPCs from marrow to peripheral blood for collection by leukapheresis. Engagement of G-CSF to its target cells indirectly decreases SDF-1a production in the marrow, allowing HPC to migrate into the peripheral blood.9 Similarly, myelosuppression by chemotherapeutic agents decreases SDF-1a levels in the marrow, thereby causing HPC mobilization into the peripheral blood.10 SDF-1a alters CD341 cell motility and up regulates surface adhesion molecules, subsequently arresting their transmigration through vascular endothelium.11 A relatively new fast-acting mobilizing agent, plerixafor (Genzyme, Boston, MA), facilitates HPC mobilization to peripheral blood by preventing the engagement of CXCR4 by its ligand SDF-1a.12 In addition, treatment of HPCs with anti-CXCR4 abolishes the engraftment capacity of the treated cells in animal models.13 SDF-1a is therefore an important chemokine that plays a central role in mobilization and homing of HPCs.
To investigate the role of CD81 T cells in HPC mobilization and engraftment, we compared the counts and gene expression levels of CD81 T cells in autologous HPC products. CD81 T and CD341 cells were purified and chemotaxis assays were performed using differing ratios of CD81 T cells to CD341 cells to assess the effect of CD81 T cells on HPC migration from high to low concentrations of SDF-1a. We then assessed the correlation of CD81 T cell to CD341 cell ratios with neutrophil engraftment.
MATERIALS AND METHODS Patient characteristics We studied 192 autologous HPC transplant patients to assess the role of CD81 T cells in HPC mobilization and engraftment. HPC collection and transplant occurred between 2003 and 2008 at the Mayo Clinic Florida. Institutional review board approval was obtained and patients were consented before the study was conducted. Patient characteristics are summarized in Table 1. The median donor age was 58.5 years (range, 18-76 years); 57.3% of the patients were males. Time to neutrophil engraftment was defined as the first of three consecutive days of an absolute neutrophil count of 500 3 106/L or higher. Similarly, time to platelet (PLT) engraftment was defined as the first of 3 consecutive days of a PLT count of 50 3 109/ L or higher.
HPC mobilization and collection All patients were first mobilized with a single daily dose of 10 mg/kg filgrastim subcutaneously for approximately 4 to 5 days. Less than 10% of the study patients received a prolonged mobilization period, some with a few weeks break, because of poor mobilization and failure to reach a peripheral CD341 cell threshold of 10/mL. None of the study patients received chemomobilization or plerixafor. On the day their peripheral CD341 cell count reached 10 3 106/L or higher, collection was initiated. Leukapheresis procedures were performed using an apheresis system (COBE Spectra, Gambro BCT, Lakewood, CO). The venous access for all autologous donors was via trilumen central venous catheter. All donors were subjected to a 240minute collection time, which resulted in an approximately 4-blood-volume collection.
Flow cytometric enumeration CD341 cell and T-cell subset counts The CD341 cell counts were performed following the ISHAGE protocol.14 Total CD341 cell yield was defined as the cumulative total number of CD341 cells from all collection days. The stem cell products were characterized by the total nucleated cell (TNC) count and total number of mononuclear cells (MNCs), CD341 cells, and T-cell Volume 55, July 2015 TRANSFUSION 1759
RUSSELL ET AL.
subsets. MNCs were stained with anti-CD45, -CD3, -CD4, and -CD8 antibodies (BD PharMingen, San Diego, CA) and anti-CD34 (Becton-Dickinson Immunocytometry Systems, San Jose, CA) and then analyzed with a flow cytometer (FACSCalibur, BD Biosciences, Waltham, MA). The number of CD341 cells was determined before cryopreservation. Data acquisition was performed using computer software (Cellquest, BD Biosciences). The percentages and absolute counts of CD341, CD31, CD31CD41, and CD31CD81 T cells were determined.
RNA extraction and real-time polymerase chain reaction RNA isolation for examining gene expression by quantitative real-time polymerase chain reaction (PCR) was performed as previously described.15 Briefly, the quantity of individual mRNAs was determined using two-step quantitative reverse transcriptase–mediated real-time PCR (qPCR). Reverse transcription of total RNA was performed using the high-capacity cDNA kit (Archive, Applied Biosystems, Carlsbad, CA). qPCR procedures were performed using universal PCR master mix (TaqMan, Applied Biosystems) and 10 ng of input cDNA for both target genes and endogenous controls. Amplification data were collected using a sequence detector (Prism 7900, Applied Biosystems) and analyzed using the sequence detection system software (Applied Biosystems). Data were normalized to 18S rRNA and mRNA abundance was calculated using the DDCT method. Qualitative PCR and qPCR were performed to assess expression levels of CD34 and CD8 mRNA. The probe set and primer pairs used for each gene are as follows: CD34 (Applied Biosystems, Catalog Number 4331182, Assay ID Hs00156373_m1; primer pairs—forward, 50 -TGAAAAAGCTGGGGATCCTAGA-30 , and reverse, 50 -TCCCAGGTCCTGAGCTATAGCC-30 ; product size, 234 bp) and CD8 (Applied Biosystems, Catalog Number 4331182, Assay ID Hs00233520_m1; primer pairs—forward, 50 -TTTCGGCGAGATACGTCTAACCCT-30 , and reverse, 50 -TTTCTCCCCCTTTGTAAAACGGGCG-30 ; product size 379 bp). For qualitative PCR, normal MNCs from high and low mobilizers and GAPDH were used as controls.
Migration studies Using a modified chemotaxis assay protocol that has been previously reported,2 we assessed the effect of CD81 Tcell depletion on HPC migration away from high concentrations of stromal cell–derived factor (SDF-1a). CD341 and CD81 T-cell populations were selected and purified using a cell isolation system (EasySep, STEMCELL Technologies, Vancouver, British Columbia, Canada). Migrations were performed using a permeable support system (Transwell, Costar, Cambridge, MA) and Transwell inserts (5 mm pore size) were coated with a confluent layer 1760 TRANSFUSION Volume 55, July 2015
of bone marrow epithelial cells (obtained from Dr D. Scadden, Harvard Medical School, Boston, MA). All chemotaxis assays were performed with only one patient product at a time. To set up the experiment, three cell fractions were generated: purified CD341 cells, purified CD81 T cells, and the negative fraction (pour off) containing all cells except CD341 and CD81 cells. Cells were seeded in triplicate in the ratios of 2:1 or 5:1 CD8:CD34 cells and a control group, which contained the pour-off cells along with CD341 cells. The control group contained all cells including CD41 cells but no CD81 cells. The negative fraction was also used for the make-up cells in the 2:1 and 5:1 groups so that each well contained the same number of cells. Purified CD341 cells were plated to the bone marrow epithelial cell–coated inserts without (no CD81) or with purified CD81 T cells at ratios of 2:1 or 5:1 (CD8:CD34) in the presence of SDF-1a concentrations of 0, 200, or 400 ng/mL. A total of 4.5 3 105 cells were plated into each Transwell insert. All wells received a constant number of 7.5 3 104 CD341 cells. A total of 1.5 3 105 CD81 cells were added to create a 2:1 ratio of CD81 to CD341 cells, and similarly, 3.75 3 105 CD81 cells were added to create a 5:1 ratio. To obtain a consistent final cell number of 4.5 3 105 cells in all wells, any remaining cells needed were obtained from the CD8– CD34– pour-off fraction from the selection procedure. The lower wells of the Transwell plate contained media only. Cells were cocultured at 37 C/5% CO2 for 4 hours, allowing migration away from higher SDF-1a concentrations into the lower wells. The migrated cells were harvested and then enumerated.
Statistical analysis The Kruskal-Wallis test was used for testing differences among the three mobilization groups. When only two categorical groups were compared, the Wilcoxon rank-sum test was used. Linear correlation between two continuous factors was assessed by Spearman’s rank correlation. As an exploratory analysis, a smoothed local linear regression technique was used to examine the relationship among increasing CD341 cell dose, CD81 T-cell dose, CD8:CD34 ratios, and days to neutrophil engraftment (TNE). No statistical adjustment was made for performing multiple tests. All statistical tests are two-sided and a p value less than or equal to 0.05 was considered significant.
RESULTS HPC product characteristics and engraftment Patients were categorized as low, moderate, or high mobilizers according to their total CD341 cell collection quartile distribution: not more than 3.7, 3.8 to 5.5, or at least 5.6 3 106/kg, respectively. HPC product characteristics and engraftment are summarized in Table 2. The number
ROLE OF CD81 T CELLS IN HPC MOBILIZATION AND ENGRAFTMENT
TABLE 2. Association of CD341 cell mobilization and HPC product counts and engraftment* Mobilization group Mobilization days Leukapheresis collections TNCs 3108/kg MNCs 3108/kg CD341 cell 3106/kg Percent CD41 T cells (%) CD41 T cells 3108/kg CD41 T:CD341 cell ratio Percent CD81 T cells (%) CD81 T cells 3108/kg CD81 T: CD341 cell ratio Lymphoma Plasma cell disease Leukemia
Low (n 5 10)
Moderate (n 5 21)
High (n 5 10)
p value
8 (6-22) 5 (2-8) 18.5 (2.3-45.2) 12.5 (1.4-44.2) 3.03 (0.4-3.7) 28 (14-54) 1.18 (0.43-5.13) 44 (17-173) 32 (13-72) 1.3 (0.4-20.5) 43 (18-693) 6 4 0
7 (2-18) 3 (1-8) 9.8 (1.5-38.3) 7.5 (1.1-30) 4.6 (3.7-5.6) 42 (10-67) 0.89 (0.01-10.5) 20 (0.2-135) 18 (3-41) 0.5 (0.002-6.7) 11 (0.1-138) 3 17 1
6 (2-22) 2 (1-7) 9.2 (1-22) 7.1 (0.3-20.2) 6.7 (5.6-18.6) 51 (3.3-66) 1.2 (0.01-10.5) 18 (0.04-151) 15 (5.2-29) 0.3 (0.01-5.1) 5 (0.1-73) 2 8 0
0.006†
Dual roles of autologous CD81 T cells in hematopoietic progenitor cell mobilization and engraftment Athena Russell,1 Sunita Malik,1 Mark Litzow,2 Dennis Gastineau,2 Vivek Roy,3 and Abba C. Zubair1
BACKGROUND: Poor marrow cellularity alone cannot explain poor hematopoietic progenitor cell (HPC) mobilization. This study assessed the role of CD81 T cells in HPC cell mobilization and engraftment. STUDY DESIGN AND METHODS: Mobilization and engraftment were assessed in 192 autologous HPC donors. CD341, CD41, and CD81 T-cell contents in apheresis products were evaluated. Using a chemotaxis assay, we assessed the effect of purified autologous CD81 T cells from low and high mobilizers on HPC migration from high to low stromal cell–derived factor (SDF-1a) concentration gradients. We also assessed CD81 T-cell content association with days to neutrophil engraftment. RESULTS: The median number of CD341 cells/kg was 4.7 3 106. Patients were categorized according to their total CD341 cell collection quartile distribution into low, moderate, and high mobilizers. We found that HPC products from low mobilizers contained more CD81 T cells than HPC products from moderate and high mobilizers. Chemotaxis assays showed depletion of CD81 T cells enhances HPC mobilization independent of SDF-1a concentration. Neutrophil engraftment analysis showed that the higher the CD81 T-cell content per unit CD341 cell, the faster the rate of engraftment. CONCLUSION: Our findings suggest CD81 T cells inhibit HPC mobilization and facilitate homing and engraftment.
F
ailure to collect adequate CD341 cell numbers following poor hematopoietic progenitor cell (HPC) mobilization is generally thought to be due to marrow damage after previous chemoand radiation therapies. Alkylating agents and high radiation dose are known to cause greater marrow damage.1 However, marrow damage alone cannot account for all poor mobilizations because there are donors who have high marrow cellularity for their age but fail to adequately mobilize enough CD341cells to achieve target cell dose. In addition, the relationship between CD341 cell dose and engraftment rate is not linear, suggesting that other factors may play a role during engraftment. Previous animal studies suggest CD81 T cells are important for CD341 cell homing to the marrow.2-5 We hypothesize that CD81 T cells have dual roles by inhibiting CD341 cell mobilization and facilitating engraftment. The stem cell compartment is composed of different types of cells whose presence is critical to stem cell physiology. These cells include mature differentiated hematopoietic cells such as T cells, dendritic cells, macrophages,
ABBREVIATIONS: qPCR 5 quantitative reverse transcriptase– mediated real-time polymerase chain reaction; SDF 5 stromal cell–derived factor; TNC 5 total nucleated cell; TNE 5 days to neutrophil engraftmen. From the 1Transfusion Medicine, Department of Pathology; and the 3Division of Hematology/Oncology, Mayo Clinic, Jacksonville, Florida; and the 2Division of Hematology/ Oncology, Mayo Clinic, Rochester, Minnesota Supported by NIH Grant CA102824. Address reprint requests to: Abba Zubair, MD, PhD, Transfusion Medicine, Department of Pathology, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL 32224; e-mail: zubair.abba@ mayo.edu. Received
for
publication
August
31,
2014;
revision
received November 20, 2014; and accepted November 28, 2014. doi:10.1111/trf.13073 C 2015 AABB V
TRANSFUSION 2015;55;1758–1765 1758 TRANSFUSION Volume 55, July 2015
ROLE OF CD81 T CELLS IN HPC MOBILIZATION AND ENGRAFTMENT
TABLE 1. Patient characteristics Mobilization CD341 cells (3106/kg) Number (192) Median age (years) Sex, female (%) Race* White Hispanic Black Other Disease categories* Leukemia Lymphoma Plasma cell disease
Low (3.7)
Moderate (3.8-5.5)
High (5.6)
44 59.5 56.8
99 59 37.4
49 55 40.8
37 2 4 1
(84) (5) (9) (2)
1 (2) 24 (55) 19 (43)
91 2 3 3
(92) (2) (3) (3)
47 (96) 0 1 (2) 1 (2)
4 (4) 22 (22) 73 (74)
0 15 (31) 34 (69)
* Data are reported as number (%).
and others. It has long been determined that stem cells require accessory cells to maintain their physiologic state.6 The role of T cells as facilitators of HPC engraftment became apparent when marrow grafts were depleted of T cells to minimize graft-versus-host disease.7 It was observed that T-cell depletion in autologous and allogeneic transplants resulted in delayed engraftment during the first 6 months after transplantation. This was further supported by clinical trials involving allogeneic transplant, which showed that the presence of CD81 T cells but not CD41 cells resulted in faster engraftment.5 Further studies in mice have also demonstrated that CD81 T cells facilitate HPC engraftment.8 The mechanism of action by which CD81 T cells facilitate allogeneic engraftment was largely attributed to elimination of residual host immune cells.5 However, it was unclear how CD81 T cells facilitated engraftment in autologous transplant settings. Granulocyte–colony-stimulating factor (G-CSF) is the principal growth factor used clinically to mobilize HPCs from marrow to peripheral blood for collection by leukapheresis. Engagement of G-CSF to its target cells indirectly decreases SDF-1a production in the marrow, allowing HPC to migrate into the peripheral blood.9 Similarly, myelosuppression by chemotherapeutic agents decreases SDF-1a levels in the marrow, thereby causing HPC mobilization into the peripheral blood.10 SDF-1a alters CD341 cell motility and up regulates surface adhesion molecules, subsequently arresting their transmigration through vascular endothelium.11 A relatively new fast-acting mobilizing agent, plerixafor (Genzyme, Boston, MA), facilitates HPC mobilization to peripheral blood by preventing the engagement of CXCR4 by its ligand SDF-1a.12 In addition, treatment of HPCs with anti-CXCR4 abolishes the engraftment capacity of the treated cells in animal models.13 SDF-1a is therefore an important chemokine that plays a central role in mobilization and homing of HPCs.
To investigate the role of CD81 T cells in HPC mobilization and engraftment, we compared the counts and gene expression levels of CD81 T cells in autologous HPC products. CD81 T and CD341 cells were purified and chemotaxis assays were performed using differing ratios of CD81 T cells to CD341 cells to assess the effect of CD81 T cells on HPC migration from high to low concentrations of SDF-1a. We then assessed the correlation of CD81 T cell to CD341 cell ratios with neutrophil engraftment.
MATERIALS AND METHODS Patient characteristics We studied 192 autologous HPC transplant patients to assess the role of CD81 T cells in HPC mobilization and engraftment. HPC collection and transplant occurred between 2003 and 2008 at the Mayo Clinic Florida. Institutional review board approval was obtained and patients were consented before the study was conducted. Patient characteristics are summarized in Table 1. The median donor age was 58.5 years (range, 18-76 years); 57.3% of the patients were males. Time to neutrophil engraftment was defined as the first of three consecutive days of an absolute neutrophil count of 500 3 106/L or higher. Similarly, time to platelet (PLT) engraftment was defined as the first of 3 consecutive days of a PLT count of 50 3 109/ L or higher.
HPC mobilization and collection All patients were first mobilized with a single daily dose of 10 mg/kg filgrastim subcutaneously for approximately 4 to 5 days. Less than 10% of the study patients received a prolonged mobilization period, some with a few weeks break, because of poor mobilization and failure to reach a peripheral CD341 cell threshold of 10/mL. None of the study patients received chemomobilization or plerixafor. On the day their peripheral CD341 cell count reached 10 3 106/L or higher, collection was initiated. Leukapheresis procedures were performed using an apheresis system (COBE Spectra, Gambro BCT, Lakewood, CO). The venous access for all autologous donors was via trilumen central venous catheter. All donors were subjected to a 240minute collection time, which resulted in an approximately 4-blood-volume collection.
Flow cytometric enumeration CD341 cell and T-cell subset counts The CD341 cell counts were performed following the ISHAGE protocol.14 Total CD341 cell yield was defined as the cumulative total number of CD341 cells from all collection days. The stem cell products were characterized by the total nucleated cell (TNC) count and total number of mononuclear cells (MNCs), CD341 cells, and T-cell Volume 55, July 2015 TRANSFUSION 1759
RUSSELL ET AL.
subsets. MNCs were stained with anti-CD45, -CD3, -CD4, and -CD8 antibodies (BD PharMingen, San Diego, CA) and anti-CD34 (Becton-Dickinson Immunocytometry Systems, San Jose, CA) and then analyzed with a flow cytometer (FACSCalibur, BD Biosciences, Waltham, MA). The number of CD341 cells was determined before cryopreservation. Data acquisition was performed using computer software (Cellquest, BD Biosciences). The percentages and absolute counts of CD341, CD31, CD31CD41, and CD31CD81 T cells were determined.
RNA extraction and real-time polymerase chain reaction RNA isolation for examining gene expression by quantitative real-time polymerase chain reaction (PCR) was performed as previously described.15 Briefly, the quantity of individual mRNAs was determined using two-step quantitative reverse transcriptase–mediated real-time PCR (qPCR). Reverse transcription of total RNA was performed using the high-capacity cDNA kit (Archive, Applied Biosystems, Carlsbad, CA). qPCR procedures were performed using universal PCR master mix (TaqMan, Applied Biosystems) and 10 ng of input cDNA for both target genes and endogenous controls. Amplification data were collected using a sequence detector (Prism 7900, Applied Biosystems) and analyzed using the sequence detection system software (Applied Biosystems). Data were normalized to 18S rRNA and mRNA abundance was calculated using the DDCT method. Qualitative PCR and qPCR were performed to assess expression levels of CD34 and CD8 mRNA. The probe set and primer pairs used for each gene are as follows: CD34 (Applied Biosystems, Catalog Number 4331182, Assay ID Hs00156373_m1; primer pairs—forward, 50 -TGAAAAAGCTGGGGATCCTAGA-30 , and reverse, 50 -TCCCAGGTCCTGAGCTATAGCC-30 ; product size, 234 bp) and CD8 (Applied Biosystems, Catalog Number 4331182, Assay ID Hs00233520_m1; primer pairs—forward, 50 -TTTCGGCGAGATACGTCTAACCCT-30 , and reverse, 50 -TTTCTCCCCCTTTGTAAAACGGGCG-30 ; product size 379 bp). For qualitative PCR, normal MNCs from high and low mobilizers and GAPDH were used as controls.
Migration studies Using a modified chemotaxis assay protocol that has been previously reported,2 we assessed the effect of CD81 Tcell depletion on HPC migration away from high concentrations of stromal cell–derived factor (SDF-1a). CD341 and CD81 T-cell populations were selected and purified using a cell isolation system (EasySep, STEMCELL Technologies, Vancouver, British Columbia, Canada). Migrations were performed using a permeable support system (Transwell, Costar, Cambridge, MA) and Transwell inserts (5 mm pore size) were coated with a confluent layer 1760 TRANSFUSION Volume 55, July 2015
of bone marrow epithelial cells (obtained from Dr D. Scadden, Harvard Medical School, Boston, MA). All chemotaxis assays were performed with only one patient product at a time. To set up the experiment, three cell fractions were generated: purified CD341 cells, purified CD81 T cells, and the negative fraction (pour off) containing all cells except CD341 and CD81 cells. Cells were seeded in triplicate in the ratios of 2:1 or 5:1 CD8:CD34 cells and a control group, which contained the pour-off cells along with CD341 cells. The control group contained all cells including CD41 cells but no CD81 cells. The negative fraction was also used for the make-up cells in the 2:1 and 5:1 groups so that each well contained the same number of cells. Purified CD341 cells were plated to the bone marrow epithelial cell–coated inserts without (no CD81) or with purified CD81 T cells at ratios of 2:1 or 5:1 (CD8:CD34) in the presence of SDF-1a concentrations of 0, 200, or 400 ng/mL. A total of 4.5 3 105 cells were plated into each Transwell insert. All wells received a constant number of 7.5 3 104 CD341 cells. A total of 1.5 3 105 CD81 cells were added to create a 2:1 ratio of CD81 to CD341 cells, and similarly, 3.75 3 105 CD81 cells were added to create a 5:1 ratio. To obtain a consistent final cell number of 4.5 3 105 cells in all wells, any remaining cells needed were obtained from the CD8– CD34– pour-off fraction from the selection procedure. The lower wells of the Transwell plate contained media only. Cells were cocultured at 37 C/5% CO2 for 4 hours, allowing migration away from higher SDF-1a concentrations into the lower wells. The migrated cells were harvested and then enumerated.
Statistical analysis The Kruskal-Wallis test was used for testing differences among the three mobilization groups. When only two categorical groups were compared, the Wilcoxon rank-sum test was used. Linear correlation between two continuous factors was assessed by Spearman’s rank correlation. As an exploratory analysis, a smoothed local linear regression technique was used to examine the relationship among increasing CD341 cell dose, CD81 T-cell dose, CD8:CD34 ratios, and days to neutrophil engraftment (TNE). No statistical adjustment was made for performing multiple tests. All statistical tests are two-sided and a p value less than or equal to 0.05 was considered significant.
RESULTS HPC product characteristics and engraftment Patients were categorized as low, moderate, or high mobilizers according to their total CD341 cell collection quartile distribution: not more than 3.7, 3.8 to 5.5, or at least 5.6 3 106/kg, respectively. HPC product characteristics and engraftment are summarized in Table 2. The number
ROLE OF CD81 T CELLS IN HPC MOBILIZATION AND ENGRAFTMENT
TABLE 2. Association of CD341 cell mobilization and HPC product counts and engraftment* Mobilization group Mobilization days Leukapheresis collections TNCs 3108/kg MNCs 3108/kg CD341 cell 3106/kg Percent CD41 T cells (%) CD41 T cells 3108/kg CD41 T:CD341 cell ratio Percent CD81 T cells (%) CD81 T cells 3108/kg CD81 T: CD341 cell ratio Lymphoma Plasma cell disease Leukemia
Low (n 5 10)
Moderate (n 5 21)
High (n 5 10)
p value
8 (6-22) 5 (2-8) 18.5 (2.3-45.2) 12.5 (1.4-44.2) 3.03 (0.4-3.7) 28 (14-54) 1.18 (0.43-5.13) 44 (17-173) 32 (13-72) 1.3 (0.4-20.5) 43 (18-693) 6 4 0
7 (2-18) 3 (1-8) 9.8 (1.5-38.3) 7.5 (1.1-30) 4.6 (3.7-5.6) 42 (10-67) 0.89 (0.01-10.5) 20 (0.2-135) 18 (3-41) 0.5 (0.002-6.7) 11 (0.1-138) 3 17 1
6 (2-22) 2 (1-7) 9.2 (1-22) 7.1 (0.3-20.2) 6.7 (5.6-18.6) 51 (3.3-66) 1.2 (0.01-10.5) 18 (0.04-151) 15 (5.2-29) 0.3 (0.01-5.1) 5 (0.1-73) 2 8 0
0.006†
