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作者：Mario Ottoa, Xiaohua Chena, William J. Martinb, Wing Leunga, James Knowlesa, Marti Holladaya, Jim Houstona, Rupert Handgretingerc, Raymond C. Barfielda作者单位：aDivision of Stem Cell Transplantation, Department of Hematology/Oncology, St. Jude Children
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【摘要】, a( Y; n" u& Y6 i% r: E
The objective of this study was to compare the patterns of T-cell differentiation from CD34 human stem cells selected with different classes of antibody targeting the CD34 molecule. We compared signal-joint T-cell receptor excision circle (sjTREC) production in thymocytes selected with different classes of anti-CD34 antibody. Based on these results, we studied immune reconstitution in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice using human stem cells selected with the same antibodies that yielded variation in the thymocytes. Human CD34 stem cells were immunomagnetically selected using the class II QBEnd antibody (prevalent in clinical graft engineering) and the class III 8G12 antibody (common in diagnostic tests). Engraftment and T-cell reconstitution were examined after transplantation. Thymocytes selected with the 8G12 class III antibody have a higher TREC production than those selected with the QBEnd class II antibody. Of mice transplanted with cells selected using the 8G12 antibody, 50% had sjTREC production, compared with 14% of mice transplanted with cells selected using the clinically common antibody QBEnd. 8G12 thymic progenitors are characterized by higher quality in thymic distribution and higher activity in T-cell differentiation. Using class III antibody targeting the CD34 molecule resulted in increased T-cell reconstitution in the NOD/SCID mouse. Use of a single antibody epitope targeting the CD34 molecule may lead to loss of cells that might provide richer T-cell reconstitution. Use of different or multiple epitopes, targeting of alternate stem cell markers, or use of cell-depletion strategies might prevent this loss. 0 u& `- [3 |% M6 E1 R0 q
【关键词】 Immune reconstitution Hematopoietic stem cell transplantation CD selection CD progenitors EngraftmentT cell Thymus Thymopoiesis
' F3 w+ |, x( p8 g INTRODUCTION, Y* h7 C. B H8 i0 y
" G+ R7 G8 p/ s7 f+ `" g. oThe CD34 molecule has long been recognized as a marker for hematopoietic stem cells (HSCs) and has been the primary target used to isolate these cells for clinical hematopoietic stem cell transplantation (HSCT). The complex CD34 antigen has been classified into three groups on the basis of the epitopes bound by monoclonal antibodies to CD34 glycophosphoprotein and the differential sensitivity of the CD34 epitopes to enzymatic cleavage by neuraminidase, chymopapain, and a glycoprotease from Pasteurella hemolytica .
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$ \( `1 G8 J! E0 AIn clinical transplantation, the QBEnd antibody to class II CD34 antigen has been used most commonly to isolate HSCs. However, other classes of anti-CD34 antibodies used in diagnostics identify populations of cells that are CD34 but are not identical to those selected using the QBEnd antibody . Because rapid T-cell reconstitution after stem cell transplantation is a crucial step for the success of this therapy modality, we were intrigued by the possibility that the use of QBEnd-CD34 antibody for stem cell enrichment might exclude important CD34 subpopulations, especially CD34 T-cell precursors that could otherwise be identified and selected by using a different antibody, such as 8G12-CD34.' N2 `4 T% E# M
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In this study, we began by looking at human thymocytes selected with antibodies targeting several different classes of CD34 antigen. Based upon these observations, we asked whether engraftment and reconstitution patterns would differ in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice that received grafts of human HSCs selected with QBEnd antibodies versus 8G12 antibodies. If engraftment, especially T-cell reconstitution, differs when different epitopes are targeted to select CD34 cells, the dominant use of the QBEnd epitope in the clinical setting should perhaps be re-evaluated: a single antibody might not only reduce the total number of stem cells collected but also miss subtypes of progenitor cells with potential for speeding immune recovery. To our knowledge, there have been no studies assessing engraftment with different classes of CD34 stem cells transplanted into experimental animals. r& K2 O! ^! }) W! C$ e4 f) O
6 C5 U" n* ?2 N7 z5 w/ I5 L9 fMATERIALS AND METHODS' C* d, H9 o5 P; e; [3 O9 H
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Acquisition and Processing of Thymocytes6 n' V' V. S) I2 [% y, l# K
8 Q7 ?) f f* g1 NWhole human thymuses discarded in the normal course of cardiac surgery on children 2 months to 10 years of age were obtained with Institutional Review Board approval (kindly provided by the Department of Surgery, LeBonheur Children's Medical Center, Memphis, TN). The tissue was placed in sterile RPMI-1640 medium (Invitrogen Corporation, Carlsbad, CA, http://www.invitrogen.com), cut into small pieces, and further processed by using a CELLECTOR Tissue Sieve System (E-C Apparatus Corporation, Holbrook, NY). The cell-rich suspension was strained through 40-µm filters. The resulting product was separated by centrifugation on a Ficoll density gradient (Ficoll-PaquePlus; GE Healthcare, Little Chalfont, Buckinghamshire, U.K., http://www.gehealthcare.com). The mononuclear cell layer was removed, washed twice with phosphate-buffered saline (PBS) (Cambrex Bio Science Walkersville, Inc., Walkersville, MD, http://www.cambrex.com), and divided into two aliquots. For QBEnd selection, cells were labeled with anti-CD34 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) and subsequently positively enriched on an AutoMACS device (Miltenyi Biotec) in accordance with the manufacturer's instructions. For 8G12 selection, mononuclear cells were incubated for 20 minutes with 4 µl of phycoerythrin (PE)-conjugated anti-CD34 antibody (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) per 1 x 106 cells at a final concentration of 1 x 108 per milliliter in labeling buffer containing PBS (Cambrex Bio Science Walkersville, Inc.) supplemented with 0.5% human serum albumin (Bayer Corp., Emeryville, CA, http://www.bayer.com) and 2 mM EDTA (Cambrex Bio Science Rockland, Inc., Rockland, ME, http://www.cambrex.com). After the incubation, the cell suspension was washed twice with labeling buffer, incubated with anti-PE microbeads (Miltenyi Biotec), and subsequently positively enriched on the AutoMACS device in accordance with the manufacturer's instructions. For some experiments, cells were further sorted by flow cytometry on a FACSVantage DiVa (BD Biosciences) to obtain the purest T-cell subset samples attainable by current methods.8 f% ?7 l; |1 @: \3 w% N ~
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Detection and Quantification of Human Signal-Joint T-Cell Receptor Excision Circle in Human and Mouse Thymus
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CD34 thymocytes from six donors were enriched with either anti-CD34 (8G12) or QBEnd antibody, as described above, and tested for signal-joint T-cell receptor excision circle (sjTREC) production. Isolated CD34 thymocytes from two donors were further sorted into CD4 and CD4¨C subpopulations on a flow cytometric sorting device (FACS Vantage DiVa; BD Biosciences) and subjected to sjTREC analysis. The sjTREC levels were quantified by real-time polymerase chain reaction (PCR) as described previously . In brief, the DNA was purified by using the QIAmp Blood Kit (Qiagen Inc., Valencia, CA, http://www1.qiagen.com). The real-time PCR was performed in a volume of 25 µl, with a final concentration of 1x Taqman Universal PCR Master Mix (Applied Biosystems, Foster City, CA, https://www2.appliedbiosystems.com), 250 nM forward primer and reverse primer, 200 nM probe, and 100 ng of DNA. The real-time PCR was performed on an ABI 7700 instrument (Applied Biosystems). All of the samples were measured in duplicate PCR reactions. The C constant region was used as an internal control and to normalize for input DNA. The standard was created by cloning the sjTREC fragment in PCR 2.1-TOPO vector using the TOPO TA Cloning Kit (Invitrogen Corporation). The standard curve and TREC values were analyzed by using ABI 7700 software. The number of TREC molecules in the sample was calculated as copies per 105 cells.
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Mobilization and Collection of Peripheral Blood Stem Cells( W. Z6 b6 O, i/ o+ \
+ y0 C. U7 y& ZPeripheral blood stem cells were mobilized in five healthy volunteer donors by administering granulocyte cell-stimulating factor at 10 µg/kg per dose (maximum dose 480 µg/day) (Neupogen; Amgen, Thousand Oaks, CA, http://www.amgen.com). A single leukapheresis was performed on day 5 by using a Cobe Spectra instrument (Cobe, Lakewood, CO, http://www.gambrobct.com). All donors gave written informed consent, and the study was approved by the St. Jude Children's Research Hospital Institutional Review Board.
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Stem Cell Enrichment from Leukapheresis Product
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To most efficiently eliminate mature T and B cells from the graft, we performed large-scale T- and B-cell depletion before stem cell selection as previously described by our group . After T- and B-cell depletion, the mean percentage of residual T cells was 0.02% (range 0.01%¨C0.04%), with a mean log10 depletion of 3.4 (range 3¨C3.8). The mean percentage of contaminating B cells was 0.1% (range 0.01%¨C0.4%), with a mean log10 depletion of 2.2 (range 1.4¨C3). 8G12 and QBEnd CD34 progenitor cells were subsequently isolated using the procedure described for the thymic CD34 progenitors.
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) B3 i1 ]$ Q/ o o! L* M0 x, mFlow Cytometry
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- q7 x4 d0 ? M9 O7 N! b, t ]) T) @Flow cytometric analysis and data analysis were performed on a BD LSR flow cytometer (BD Biosciences). Expression of human leukocyte surface markers was determined using antibodies labeled with fluorescein isothiocyanate, PE, PE-cyanine 7 (PE-Cy7), PE-Cy5, allophycocyanin, or PE-Texas Red targeting the following antigens: CD3, CD4, CD13, CD19, and CD34-QBEnd10 (Dako North America, Inc., Carpinteria, CA, http://www.dakousa.com); CD20, CD34-581, and CD45-streptavidin (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com); pan-T-cell receptor (TCR)- and CD34-8G12 (BD Biosciences); CD7 pan-TCR-ß (Caltag Laboratories, now part of Invitrogen Corporation); CD34-AC136 (Miltenyi Biotec); and CD34-12.8 (Baxter, Deerfield, IL, https://www.baxter.com). We stained for the mouse leukocyte common antigen with anti-mouse CD45 (BD Biosciences). Cells were stained according to the manufacturers' protocols. When necessary, red blood cells were lysed with 1x ammonium chloride solution. Viability was assessed by propidium iodide staining (Roche Diagnostics, Manheim, Germany, http://www.roche.de).
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: n5 z( c$ C) ]: ?5 `Transplantation of NOD/SCID Mice) `- K) [ h6 Q" ]6 [
! M4 M+ n( g% K4 I# Y( YNOD/SCID mice (NOD/LtSz-Prkdcscid/J) cared for according to institutional guidelines were sublethally irradiated with 325 cGy by using a 137Cs source (J L Shepherd and Associates, San Fernando, CA, http://www.jlshepherd.com). Two groups of mice received transplants of 5 x 105 HSC selected with either QBEnd (n = 69) or 8G12 (n = 44). In addition to the mice transplanted with the stem cell groups of interest, we transplanted mice with nonselected cells from the T/B-depleted product, targeting the same number of CD34 stem cells in the grafts in order to compare engraftment and sjTREC production (n = 47). The cells were injected into the lateral tail vein. Half of the mice were humanely sacrificed after 10¨C12 weeks and half after 6 months. Bone marrow was harvested by flushing the femora and tibiae. Spleen and thymus tissues were processed to obtain single-cell suspensions.! i' E$ F1 ^6 g7 J [6 l: y
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Statistical Analyses# d. d5 u- ?$ G5 [. |0 X$ f
$ V. {: O; |+ e0 s' jFor statistical evaluation, the paired Student's t test was used. A p value less than .05 was considered to indicate statistical significance.# W* @( P% B. }" d& j6 i& z9 t
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RESULTS! H: I v9 X& D/ f2 [1 i' X s
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Distribution of Class II and Class III Epitopes of CD34 Antigen in Human Thymus9 x/ N$ G; \# B
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To understand the distribution of CD34 class III epitope in thymus, we collected 13 thymuses and examined the proportion of class III and class II epitopes on thymocytes by flow cytometry. We found that both QBEnd-class II and 8G12-class III epitopes are expressed on thymocytes. The average percentage for 8G12-class III population was higher (p = .0015) than for QBEnd-class II population. (Table 1; Fig. 1).5 x0 M2 R0 } f1 e
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Table 1. Flow cytometric analysis of thymocytes from 13 different human thymuses* H8 y3 b% }9 ^. F& Y: `' w) I4 n* M
6 W0 K8 \! j5 vFigure 1. Variations in expression of CD34 antigen in human thymocytes. The x-axis shows fluorescence density of CD7. The y-axis represents the fluorescence density for each CD34 antibody, including (A) CD34-class III-12.8, (B) CD34-class II-QBEnd, (C) CD34-class III-8G12, and (D) CD34-class III-581. Abbreviations: APC, allophycocyanin; Cy5, cyanine 5; ECD, phycoerythrin-Texas Red; FITC, fluorescein isothiocyanate; SA ECD, streptavidin phycoerythrin-Texas Red.; G" n5 C! y a7 a, O$ H [* o
" r/ E, {7 t. T( ^+ T6 TsjTREC Production in Class II or Class III Progenitor Cells in Thymus
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To test T-cell differentiation of class II and class III progenitor cells in thymus, we examined sjTREC production in these two populations of thymocytes. The mean sjTREC production in 8G12-class III thymocytes was 1,161 ¡À 774 (¡À SD) and was 514 ¡À 297 in QBEnd-class II thymocytes (p = .028; Table 2). In two thymuses, we separated the thymocytes into CD34 CD4 and CD34 CD4¨C in each CD34 epitope. The sjTREC level was very low in both CD34 CD4¨C populations. However, sjTREC production in 8G12-CD34 CD4 cells was 2.5-fold of that in QBEnd-CD34 CD4 cells in both donors (Table 3).
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9 z/ I! I+ p& c5 iTable 2. sjTREC production in thymocytes
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Table 3. sjTREC production in thymocytes sorted by flow cytometry5 U2 Y+ L7 t0 J
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Engraftment of Human CD34 Cells in NOD/SCID Mice! D# b6 k$ G! Z" U
+ g2 j' a" M5 R3 u& ATo estimate the engraftment of human CD34 cells in the mice, we used anti-human CD45 antibody to recognize human CD34 cells and lymphocytes in the mice at 10 weeks, 3 months, and 6 months after the transplantation. We collected the cells from three locations, including spleen, bone marrow, and residual thymus. We found no statistically significant difference in human CD45 engraftment from any compartment (Fig. 2, showing human CD45 engraftment from bone marrow). Mean engraftment for the CD34 QBEnd group was 18% at 3 months (n = 30) and 5.4% at 6 months (n = 28). Mean engraftment for the CD34 8G12 group was 9.7% at 3 months (n = 29) and 3.66% at 6 months (n = 18). Mean engraftment for the T/B-depleted group with no stem cell enrichment was 32% at 3 months (n = 20) and 24% at 6 months (n = 6). Using flow cytometry, we found no differences in human CD19 B-cell development or in human CD56 natural killer (NK) cell engraftment (data not shown). There were no mature T cells detectable by flow cytometry using anti-CD3 antibodies.
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( @ K( V1 T; ~4 @" LFigure 2. Human CD45 engraftment in the bone marrow of nonobese diabetic/severe combined immunodeficient mice. The y-axis shows the percentage of human CD45 cells in the mice transplanted with T/B dep cells, and cells selected with either the QBEnd antibody or the 8G12 antibody at 3 and 6 months after transplantation are described on the x-axis. represents the samples tested. The mean and median CD45 percentages for each group are represented as thick bars or thin bars, respectively. Abbreviations: mo, months; T/B dep, T/B-depleted.5 V9 O) r6 r$ s* F4 D* F
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Human sjTREC Production in Mice+ E# h: z: f& O- b8 Q% U
+ N( }; u# C* r6 s m2 s) \SjTREC is an episomal excision product formed at the point of TCR- deletion in the early stages of TCR-ß formation in thymus. Because the environment of mice thymus may not be suitable for the formation of complete human TCR-CD3 complex (targeted by the antibodies used in flow cytometry to detect CD3 T cells), we used sjTREC as a marker for detection of the early stages of human T-cell formation. We examined human sjTREC production in mouse bone marrow, spleen, and thymus at 10 weeks, 3 months, and 6 months post-transplantation. There was no detectable sjTREC in the spleen and bone marrow at any time points for the mice transplanted either by 8G12 or QBEnd progenitor cells. There was also no detectable sjTREC in their thymus at 10 weeks after the graft. However, at 3 months, 3 of 27 (11%) mice from both groups had sjTREC detectable in the thymus. At 6 months, 5 of 10 mice (50%) in the 8G12 group and 3 of 22 (14%) in the QBEnd group had detectable sjTREC in the thymus. In the mice transplanted with grafts that were T- and B-cell-depleted, but not stem cell-selected, TRECs were detected as early as 10 weeks in 3 of 11 mice (27%). The production declined over time to 10% by 6 months (Table 4).2 Y, ~4 d* Q k6 v& U
4 q, t% a7 M8 @1 o" zTable 4. Signal-joint T-cell receptor excision circle production in the thymuses of nonobese diabetic/severe combined immunodeficient mice; F- Q% `, X9 A" v& A7 K8 ~
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DISCUSSION% A% B9 |4 Z3 b
2 r8 B" S0 t' p( b l( F, RSince the 1980s, the CD34 molecule has become very important in clinical stem cell transplantation .* i Y4 w# n V
* Y7 d4 ?* N- d: a% fWe began this study because of our interest in improving thymus-dependent T-cell reconstitution in patients transplanted with CD34-selected stem cell grafts. When immunomagnetic beads are used for cell selection, the antigen specificity of the antibody that is used will affect cell selection. Given this and given the variation in the CD34 molecule, we looked at human thymocytes and evaluated the CD34 expression using different classes of anti-CD34 antibodies, namely 8G12 (class III) and QBEnd (class II). We found a clear and statistically significant difference in the ability of 8G12 antibody to identify CD34 expression in human thymocytes in comparison with QBEnd (p = .001; Table 1). This finding suggests that the 8G12 population may contain quan
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