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作者：Paul J. Shaughnessya, Carlos Bachiera, Charles F. LeMaistrea, Cagla Akayb, Brad H. Pollockb, Yair Gazittb作者单位：aTexas Transplant Institute, San Antonio, Texas, USA;bUniversity of Texas Health Science Center at San Antonio, San Antonio, Texas, USA 3 E2 B' D) Z m% b! U7 ?& _
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【摘要】
9 ~. I: H; A+ K; h1 p Dendritic cells (DCs) are effective antigen-presenting cells. We hypothesized that increasing the DC populations in donor lymphocyte infusions (DLIs) may augment the graft versus malignancy effect, particularly if granulocyte-macrophage colony-stimulating factor (GM-CSF) mobilization resulted in increased precursor dendritic cell (pDC) 1 cells. Mature DCs, pDC1 cells, pDC2 cells, and CD34 cells from the same donor were compared after granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cell collections and GM-CSF mobilized DLI collections. Mobilization with G-CSF resulted in up to a 10-fold larger number of CD34 cells per kg and a 3¨C5-fold larger number of mature DCs, pDC1 cells, and pDC2 cells within the same donor compared with GM-CSF. The ratio of pDC1 to pDC2 in each donor remained constant with either cytokine. In this small sample of normal donors, it appears that G-CSF mobilizes more CD34 cells, mature DCs, pDC1 cells, and pDC2 cells within the same donor than does GM-CSF, with no significant polarization by G-CSF or GM-CSF for either pDC1 or pDC2 cells.
% Y2 p, b4 K% i 【关键词】 Granulocyte colony-stimulating factor Granulocyte-macrophage colony-stimulating factor Graft versus host disease Dentritic cells Mobilization* s( R! M: Z3 G) N
INTRODUCTION
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5 {9 D" A5 T# fImmune modulation of the graft versus host disease (GVHD) and graft versus malignancy (GVM) effects are important but poorly understood components of allogeneic blood and marrow transplantation (ABMT). The GVM effect has been well demonstrated through the successful treatment of relapsed chronic myelogenous leukemia with donor lymphocyte infusions (DLIs) . Nevertheless, relapse of malignancy remains a major concern after ABMT, and being able to understand the mechanism and augment the response of the immunologically mediated GVM effect may improve transplant outcomes. Although the exact mechanism of GVM is not known, it is believed to be analogous to the pathophysiology of GVHD, in that alloreactive donor-derived T-cells recognize cells bearing host antigen and destroy them.! v4 z' g. @2 O
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The immunological profile of cell types, including dendritic cells (DCs), mobilized by granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) has been fully characterized .
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Recently, precursor dendritic cells (pDC) subtypes capable of generating Th1 helper T-cells (pDC1) and Th2 helper T-cells (pDC2) were described with a phenotype of CD11c human leukocyte antigen-DR (HLA-DR) lin¨C cells for pDC1 and a phenotype of interleukin-3 (IL-3) R /HLA-DR lin¨C cells for pDC2 .8 a' m. C" |: h& e- {" i' \
2 ~9 G8 \5 N j7 L* j8 CDonor lymphocytes may be collected from the same donor of an ABMT patient when relapse or incomplete donor chimerism occurs in that patient. Lymphocytes (CD3 cells) circulate in the peripheral blood of normal donors in adequate amounts such that sufficient donor lymphocytes can be collected with apheresis without any cytokine stimulation. However, we hypothesized that if normal donors were given GM-CSF prior to DLI collection, it could result in polarization of DCs toward the pDC1 phenotype and augment the GVM effect, without impacting the total lymphocyte collection or donor and patient safety. We report here the results of a pilot study of DC mobilization with GM-CSF prior to DLI collection from normal allogeneic donors compared with the same donor¡¯s peripheral blood stem cell mobilization product using G-CSF for ABMT. Preliminary results of this work have been presented previously .
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MATERIALS AND METHODS
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Donor and Patient Population3 p% q$ x9 w3 `0 L& y" \# G
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The study was approved by the investigational review board at Methodist Hospital and the University of Texas Health Science Center at San Antonio, San Antonio, TX. All patients and donors signed investigational review board-approved consent forms.7 J3 a! [1 w y$ y" i& Y
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Inclusion criteria for the study required patients and donors to be between the ages of 17 and 70 and required the patients to have relapsed malignant disease after ABMT with no active grade III or IV acute GVHD or extensive chronic GVHD. The donor must have been the original donor of the allogeneic peripheral blood stem cells and have been available for DLI collection.
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Sample Collection and Analysis
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All donors received GM-CSF 250 µg/m2 for 5 days prior to DLI collection. Peripheral blood was collected on all donors prior to starting GM-CSF and on day 5 after receiving the last dose of GM-CSF. Donor lymphocytes were collected by standard apheresis techniques. Banked samples from the original G-CSF-mobilized (G-CSF, 10 µg/kg daily for 5 days) peripheral blood stem cell (PBSC) apheresis products that were cryopreserved at the time of PBSC apheresis harvest were analyzed in comparison with the GM-CSF-mobilized DLI apheresis product.) r! m& s. Z$ x+ y
3 U6 ~0 A" `3 L+ [3 gSamples were immediately processed to isolate the mononuclear cell fraction by the Ficoll gradient method and frozen at ¨C80¡ãC as described before .
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Staining for Mature DCs and pDC1 and pDC2 Subsets7 h: I6 r2 \3 q
- t, _8 X4 y: u% j9 K5 J* i/ qStaining for mature DCs (CD80 CD14¨C cells) was performed by two-color assay using CD80-FITC and CD14-PE. Staining for pDC1 (CD11c HLA-DR lin¨C cells) was performed by three-color assay using CD11c-PE, HLA-DR-APC, and lineage markers (CD14/CD2/CD20/CD56)-FITC. Staining for pDC2 (IL-3 R /HLA-DR lin¨C cells) was performed by three-color assay using anti-CD123-PE, anti-HLA-DR-APC, and lineage markers (CD14/CD2/CD20/CD56)-FITC. All antibodies were from Becton, Dickinson and Company (San Jose, CA, http://www.bd.com). Cell fluorescence was detected by flow cytometry (FACSCalibur), and quantitation was done by the CellQuest software. Isotype matching immunoglobulin controls were run for each fluorophore.0 `; E' U- x" a5 l, k5 x
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One hundred thousand cells were analyzed. An example of staining for pDC1 and pDC2 cells is depicted in Figure 1. In this example, 79.3% of the HLA-DR /lin¨C cells were pDC1 and 47.3% were pDC2 cells.# N L7 F" I2 {8 y3 B
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Figure 1. Example of staining for precursor dendritic cell (pDC) 1 and pDC2 subsets. Immunofluorescence staining of frozen and thawed mononuclear cells was performed by three-color staining (phycoerythrin/fluorescein isothiocyanate/allophycocyanin . Staining for mature DCs (CD80 CD14¨C cells) was performed by two-color assay using CD80-FITC and CD14-PE. Staining for pDC1 (CD11c HLA-DR lin¨C cells) was performed by three-color assay using CD11c-PE, HLA-DR-APC, and lineage markers (CD14/CD2/CD20/CD56)-FITC. Staining for pDC2 (IL-3 R /HLA-DR lin¨C cells) was performed by three-color assay using anti-CD123-PE, anti-HLA-DR-APC, and lineage markers. Cells were gated first on lin¨C/HLA-DR cell population (A), and cells in this region (R1) were interrogated for CD11c (pDC1cells) (B) or CD123 DC2 cells (C). The histograms (D¨CF) represent the actual percentage of pDC1 (E) or pDC2 CELLS (F) in the R1 region above the immunoglobulin control (D). Fifty thousand cells were analyzed. In these examples, the donor had 80% pDC1 and 47% pDC2 cells. Further details are given in Materials and Methods. Abbreviations: DC, dendritic cell; HLA-DR, human leukocyte antigen-DR.0 s) x' |* s8 [
) J. M1 Z9 N+ Q* M" u+ ?4 u1 aToxicity Grading; R" O7 ]$ U8 R8 _6 M$ X' h
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Toxicity was graded by Common Terminology Criteria version 3.0 .% V0 D/ L2 ~& k( q# O- E
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Statistical Analyses
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Simple descriptive statistics were used to characterize the distribution of cell types. Comparison of the cell types determined by flow cytometry between the G-CSF-mobilized and GM-CSF-mobilized apheresis products were performed using two-sample independent t tests with adjustment for unequal variance across groups. Comparison of the yield of mature DCs per µl in peripheral blood samples and DC subsets per kg from apheresis products were performed by Student¡¯s t test (nonparametric, Mann Whitney, two-tailed) using GraphPad Prism as described previously .
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Eight patient and donor pairs were treated in the study; their characteristics are described in Table 1. All donors were HLA-matched siblings, and seven donors had samples from their prior G-CSF-mobilized PBSC harvest available for comparison with the GM-CSF-mobilized DLI collection. No serious adverse events occurred in the donors during the GM-CSF administration and apheresis procedure. All donors described mild bone pain that did not restrict activities of daily living, and pain was controlled by acetaminophen.- G# H0 \2 O) \; u( [1 K+ I- E
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Table 1. Patient and donor characteristics5 r" ~: I. `% @: S: p( z
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All patients received all or part of the DLI collection from their respective donors. No unexpected adverse events occurred during the infusion of the apheresis products from the DLI collections. Three patients developed grade II acute GVHD, and one patient developed grade IV acute GVHD. Three patients achieved remissions of their underlying malignancy post-DLI for 1¨C6 months. Subsequently, six patients died of progressive malignancy, and two patients died of sepsis with no discernable GVHD or malignancy (Table 2).
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Table 2. Patients¡¯ outcome" X9 u9 t+ E' N9 C1 o
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Mobilization of mature CD34 cells in the blood and in the apheresis products were determined for each donor mobilized with G-CSF or GM-CSF. The results are shown in Table 3. Mobilization with G-CSF resulted in 15-fold higher levels of blood CD34 cells (102 ¡À 52.7 vs. 6.5 ¡À 3.6 cells per µl) and 14-fold higher levels of CD34 cells (1,415 ¡À 1,059 vs. 119.4 ¡À 71.6) in apheresis products (Table 3).$ _" T) G1 {8 V. ?" ?- ]
. g% b9 v% m2 P( Q. yTable 3. Mobilization of CD34 HSCs with G-CSF or GM-CSF in blood and in apheresis products of same donor
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" H* I; ?3 Z1 n( t/ [Mobilization of CD3 T-cells in the apheresis products were determined for each donor mobilized with G-CSF or GM-CSF. The results are shown in Table 4. Mobilization with G-CSF resulted in a mean of 30 ¡À 8.3 x 109 cells per apheresis product, whereas mobilization with GM-CSF for DLI collections resulted in a mean of 10.3 ¡À 4.4 x 109 cells per apheresis product. Although lower levels of CD3 cells were collected in the DLI phase, the number of CD3 cells infused per kg of recipient was >108 cells per kg, sufficient for generation of T-cell responses in the recipient patient (Table 4).5 q4 j+ Y5 A K* m8 a; w% U
( o+ |; Y$ \/ p8 A3 Y3 O( qTable 4. Mobilization of CD3 T-cells with G-CSF or GM-CSF in apheresis product of same donor- ?! C9 t# t- Y2 r( k4 V2 C8 p( t
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The extent of mobilization of mature CD80 DCs, pDC1 and pDC2 cells, and pDC1/DC2 ratios in the blood and in the apheresis products were determined for each donor mobilized with G-CSF or GM-CSF. The results are shown in Table 5for G-CSF and in Table 6for GM-CSF. Mobilization with G-CSF resulted in a 10- to 20-fold increase in blood CD80 pDC1 and pDC2 DCs per µl (Table 5), whereas mobilization of the same donor with GM-CSF resulted in a much more modest increase in blood DCs per µl and in the absolute amount of DCs in the apheresis products compared with G-CSF (Tables 5, 6), although great heterogeneity was observed between individual donors, both at baseline and after mobilization with G-CSF or GM-CSF (compare Tables 5¨C7). Interestingly, pDC1/DC2 ratio was generally increased in blood and in the apheresis products after mobilization with either G-CSF or with GM-CSF, but no statistically significant skewing of DCs to pDC1 was observed (compare Tables 5¨C7).$ K* ^! {0 r5 d8 w8 R
* O% P0 Y/ S! R4 s5 wTable 5. Mobilization of pDC1, pDC2, and CD80 (mature DCs) with G-CSF in peripheral blood hematopoietic stem cell collections
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Table 6. Mobilization of pDC1, pDC2, and CD80 (mature DCs) with GM-CSF in DLI collections
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Table 7. Comparison of T-cell, HSC, and dendritic cell mobilization in the apheresis products of same donor mobilized with G-CSF versus GM-CSF
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) Q8 f, @8 ?" D/ hA summary of the results obtained from analysis of CD3, CD34, mature CD80 DCs, pDC1, and pDC2 cells per kg in the apheresis products collected after G-CSF or GM-CSF is given in Table 7. Here the values were computed per kg of recipient. Thus, mobilization with G-CSF resulted in approximately threefold higher numbers of CD3 T-cells (p = .007), whereas up to a 10-fold higher number of CD34 cells (p = .0008) was infused in recipient patients. Mobilization with G-CSF resulted also in a 2- to 4-fold larger number of mature CD80 DCs (p = .014), pDC1 cells (p = .019), and pDC2 cells (p = .023) compared with GM-CSF within the same donor (Table 7). The ratio of pDC1 to pDC2 in each donor did not vary significantly between the G-CSF-mobilized and GM-CSF-mobilized apheresis products (Table 7). The total numbers of mature CD80 DCs per kg and pDC1 pDC2 cells per kg infused to the recipient patients was much higher (>7-fold) in the transplant phase (G-CSF mobilization) compared with the DLI phase (GM-CSF mobilization) of treatment.
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2 h# Z9 J4 A4 n- lThe number of pDC1 and pDC2 cells per µl of donor¡¯s blood on day 5 mobilization with G-CSF or GM-CSF compared with baseline (day 1) is depicted in Figures 2and 3, respectively. A 10- to 100-fold increase in blood pDC1 and pDC2 cell mobilization by G-CSF was observed, both for pDC1 and pDC2 cells; however, the pDC1/pDC2 ratio in blood and in the apheresis product did not change in G-CSF-mobilized donors (compare also results in Tables 5, 7). A similar trend was observed in GM-CSF mobilization, with a 2- to 10-fold increase in pDC1 and pDC2 cells observed; however, the absolute numbers of DCs per µl in day 5 of mobilization were 10-fold lower compared with G-CSF. Most importantly, as was the case with G-CSF, the pDC1/DC2 ratio in blood and in apheresis products did not change significantly (compare also with Tables 6, 7).
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Figure 2. Comparison between day 1 and day 5 G-CSF-mobilized blood precursor dendritic cell (pDC) 1 and pDC2. Data were taken from Table 5. Only five donors were available for analysis of pDC1 and pDC2 cells at baseline in the G-CSF mobilization phase. Further details are given in Materials and Methods. Note 50- to 100-fold increase in blood pDC1 and pDC2 cell mobilization by G-CSF. Abbreviations: DC, dendritic cell; G-CSF, granulocyte colony-stimulating factor.! o& m9 t6 h* z: i9 Q
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Figure 3. Comparison between day 1 and day 5 GM-CSF mobilized blood precursor dendritic cell (pDC) 1 and pDC2. Data were taken from Table 6. Further details are given in Materials and Methods. Note 2- to 10-fold increase in blood pDC1 and pDC2 cell mobilization by GM-CSF. Abbreviations: DC, dendritic cell; GM-CSF, granulocyte-macrophage colony-stimulating factor.; ^7 }& Z4 P5 K$ A
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The results obtained from comparing mobilization of CD80 CD14¨C mature DCs in the blood of donors mobilized with G-CSF or GM-CSF are depicted in Figure 4. As was the case for pDC1 and pDC2 cells, G-CSF mobilized a greater number (10- to 50-fold) of CD80 mature DCs than GM-CSF within the same donor, whereas GM-CSF was less effective (10-fold lower) in mobilizing CD80 DCs (compare also with Tables 5¨C7). These results, taken together, suggest that G-CSF mobilized greater numbers of DCs and DC subsets; however, no skewing toward pDC1 or pDC2 was observed with either mobilizing growth factor.: k6 ^" a9 D! C4 I9 S3 e
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Figure 4. Peripheral blood mobilization of mature CD80 dendritic cells (DCs) by G-CSF blood and marrow transplantation harvest) and GM-CSF (donor lymphocyte infusion) in day 1 and day 5 of mobilization. Blood samples from donors on day 1 and day 5 were compared for the number of mature CD80 CD14-DCs. Data were taken from Tables 5 and 6. Mature CD80 DCs were defined as CD14-CD80 cells. Same donors were used for G-CSF and GM-CSF mobilization. Note a fivefold increase in CD80 cells with GM-CSF, compared with a 50- to 100-fold increase by G-CSF. Further details are given in Materials and Methods. Abbreviations: G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor. B! n! C8 w5 y" P/ _; {
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DISCUSSION
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% E7 q5 Y; a% m9 \This small study is the first to report data on G-CSF and GM-CSF as single agents used separately in the same normal donor to mobilize apheresis products. Our results are consistent with prior studies that have shown that G-CSF alone can mobilize significantly more CD34 cells than GM-CSF alone . However, we did not observe any evidence of polarization of pDC1 or pDC2 by either cytokine in the apheresis products.
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, \% ~7 T& R% a. }& t' GIt has been shown that G-CSF mobilizes more nucleated cells, most of which are granulocytes; whereas GM-CSF mobilizes fewer nucleated cells and a higher percentage of CD14 monocytes . The current study demonstrated significantly more DCs in the G-CSF-mobilized apheresis product than the GM-CSF apheresis product, which may be a result of the small number of patients in this study, using GM-CSF as a single agent, or that normal donors may respond differently to cytokine mobilization of DCs than do patients with malignancies or who have received previous cytotoxic chemotherapy. Yet another difference is that although standard doses of GM-CSF were used (250 µg/m2) in this study, GM-CSF was given for 5 days only compared with 10¨C15 days in other studies.$ I3 I, J: D* V: k) u, m
3 k" m7 Y3 n) L6 C) L1 `We and others have studied the polarizing effects of cytokines on DC subsets in normal allo-donors and in patients undergoing HSC collection for autologous transplantation . They found no significant differences in the apheresis products between the groups in CD34 or T-cell content. However, they did find significantly fewer pDC2 cells and similar numbers of pDC1 cells in the apheresis products from patients who received the combination of GM-CSF and G-CSF compared with patients who received G-CSF alone. They also noted in a separate cohort of patients who received chemotherapy followed by G-CSF and by GM-CSF, 6 days later, that there were significantly fewer pDC2 cells in the apheresis product compared with patients who received chemotherapy and G-CSF alone. These results suggest that combinations of GM-CSF with G-CSF can reduce the content of pDC2 type DCs in the apheresis product. The patients in our study did not show any polarization to either pDC1 or pDC2 phenotype with either cytokine. These results may represent the ability of normal donors to respond differently to cytokines compared with patients who have received prior cytotoxic chemotherapy. Although the patient numbers are small, Figure 2 suggests that there was only a small increase in the total numbers of CD80 cells per µl mobilization from day 1 to day 5 in the GM-CSF group, whereas in the G-CSF group, a significantly larger increase in the number of CD80 cells per µl from day 1 to day 5 was observed. The more modest mobilization of CD80 mature DCs by GM-CSF in this study could be the result of the short exposure of the donors to GM-CSF (5 days) compared with the longer schedules (10¨C15 days) used for hematopoietic stem cell mobilization. Also, the donors in our study received the G-CSF first for mobilization of stem cells prior to transplant, and then received GM-CSF prior to mobilization of DLI several weeks to months later; hence, it is possible that the use of G-CSF and GM-CSF in close proximity to each other may have different effects on dendritic cell subsets than when only G-CSF or GM-CSF are used within the same donor.- d4 X( ^4 Y/ A
$ {8 e# {7 i n' [! Y u7 z0 pIt should be noted that we did not perform parallel experiments with donors mobilized with GM-CSF first and than with G-CSF because in the context of allotransplatation we believe that G-CSF is superior in mobilization of hematopoietic stem cells for ASCT. However, lack of such a sequence of mobilization in our experimental design should not detract from the validity of out conclusions within our study.7 ?2 |) j; w/ d; t
& ]; F+ c- G$ n) bFinally, the small and heterogeneous population of patients in this pilot study precludes making any clinical determinations. However, the fact that acute GVHD was seen in some patients after DLI supports the observation that adequate alloreactive lymphocytes were collected from the donors after the GM-CSF administration. Our results with GM-CSF regarding collection of CD3 T-cells for DLI are comparable to the results published by Arat et al. using doses of 5 and 10 µg/kg G-CSF.
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2 ~3 P6 P; i8 _8 k, @6 N9 @2 IIn conclusion, we observed significantly more mature and pDCs mobilized by G-CSF than with GM-CSF with no polarization of DCs to pDC1 or pDC2 in apheresis products mobilized from normal donors. Larger studies in a more homogenous population of donors and patients may be necessary to confirm these findings. Z, E; `9 V" |- u; T3 q
: X* z+ n6 O. nDISCLOSURES
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0 h6 X2 X2 O/ JThe authors indicate no potential conflicts of interest.! c5 q+ q: Y' k* p
% V3 I% n8 k" ~ z2 c& KACKNOWLEDGMENTS
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/ n2 ~) J% F! T. ZThis study was supported in part by research funding from Berlex Corporation. We thank Charles Thomas III and the Institutional Flow Cytometry Core for performing the flow cytometry analyses.
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