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Umbilical cord blood–naive T cells but not adult blood–naive T cells require HLA class II on antigen-presenting cells for allo-immune activation
Umbilical Cord Blood–Naive T Cells But Not Adult Blood–Naive T Cells Require HLA Class II on Antigen-Presenting Cells for Allo-Immune Activation F.M. Kloosterboer, S.A.P. van Luxemburg-Heijs, R. Willemze, and J.H.F Falkenburg ABSTRACT: Because a relatively low incidence and severity of graft-versus-host disease after umbilical cord blood (UCB) transplantation is observed, we investigated whether T cells from UCB or adult blood (AB) were differentially activated by antigen-presenting cells with or without human leukocyte antigen (HLA)–DR expression. T cells from UCB or AB, or CD45RA⫹ naive T cells and CD45RO⫹ memory T cells separated from AB, were stimulated with the HLA-DR⫹ or HLA-DR⫺ cell line AML193. On days 1–3 after stimulation, numbers of interleukin (IL)–2, IL-4, IL-10 or interferon gamma (IFN␥)–secreting cells were determined by enzyme-linked immunospot analysis. No IL-4 or IL-10 was produced. AML193-DR⫹ cells induced IL-2 and IFN-␥ secretion with slower kinetics and lower levels in UCB T cells than in AB T cells. AML193-DR⫹ cells induced comparable ABBREVIATIONS AB adult blood APC antigen-presenting cell DC dendritic cell ELISPOT enzyme-linked immunospot GVHD graft-versus-host disease
INTRODUCTION Allogeneic stem cell transplantation (SCT) has been successfully used for the treatment of various hematologic and nonhematologic diseases. One of the major complications that can occur after allogeneic SCT that uses stem cell grafts from adult donors is graft-versus-host disease
From the Department of Hematology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. Address reprint requests to: Dr. F.M. Kloosterboer, Department of Hematology, Leiden University Medical Center, C2-R, P.O. Box 9600, 2300 RC Leiden, The Netherlands; Tel: ⫹31 71 526 2271; Fax ⫹31 71 526 6755; E-mail: [email protected]. Received October 6, 2003; revised December 30, 2003; accepted January 5, 2004. Human Immunology 65, 328 –339 (2004) © American Society for Histocompatibility and Immunogenetics, 2004 Published by Elsevier Inc.
IL-2 but higher IFN-␥ secretion in CD45RA⫹ T cells from AB than in UCB T cells. AML193-DR⫺ cells did not induce IL-2– or IFN-␥ secretion in UCB T cells, but stimulated both CD45RA⫹ and CD45RO⫹ T cells from AB to secrete IL-2 and IFN-␥. Thus, not only the absence of memory T cells but also the inability to respond to HLA-DR–negative antigen-presenting cells and the slower kinetics and level of activation found for naive T cells from UCB as compared with AB may partly explain the reduced antirecipient reactivity after UCB transplantation. Human Immunology 65, 328 –339 (2004). © American Society for Histocompatibility and Immunogenetics, 2004. Published by Elsevier Inc. KEYWORDS: umbilical cord blood; graft-versus-host disease; alloreactivity; HLA class II; T cells
IFN-␥ IL PBMC SCT UCB
interferon-gamma interleukin peripheral blood mononuclear cells stem cell transplantation umbilical cord blood
(GVHD). In contrast, when umbilical cord blood (UCB) is used as stem cell source for SCT, a relatively low incidence and severity of GVHD has been found in both human leukocyte antigen (HLA) identical and partially matched unrelated UCB transplants [1–3]. A diminished alloreactivity in UCB T cells has been proposed as a possible mechanism responsible for the lower incidence and severity of GVHD after UCB transplantation. Some investigators have described diminished proliferative or cytotoxic responses of UCB T cells as compared with adult blood (AB) T cells in mixed lymphocyte reaction or in limiting dilution analysis measuring the frequencies of antirecipient helper T precursor 0198-8859/04/$–see front matter doi:10.1016/j.humimm.2004.01.003
Cord Blood T Cells Need HLA-DR for Allo-Activation
cells and cytotoxic T precursor cells [4 –10]. However, others have reported similar proliferative or cytotoxic capacity for UCB T cells and AB T cells in response to allogeneic activation [4, 6, 10 –12]. Allogeneic activation of CD4⫹ T cells and CD8⫹ T cells requires T cell receptor (TCR) signaling mediated through interaction with HLA class I or HLA class II molecules, respectively, on the antigen-presenting cells (APC). Whereas nearly all cells express HLA class I, constitutive expression of HLA class II is mainly limited to professional APC, like dendritic cells (DC), except for expression on hematopoietic progenitor cells. Expression of HLA class II molecules can, however, be induced by proinflammatory cytokines, such as interferon gamma (IFN-␥) or tumor necrosis factor alpha. It has been demonstrated that CD4⫹ helper activity is required for the generation of CD8⫹ T cell responses, and as a consequence the expression of MHC class II on the APC may be essential for the activation of both CD4⫹ T cells and CD8⫹ T cells. Effective T cell activation is further facilitated by additional signaling through costimulatory and adhesion molecules that can be expressed on the APC [13–17]. The most important costimulatory molecules are CD40, which can interact with CD40 ligand on the T cell, and CD80 or CD86, which can interact with CD28 on the T cell. The adhesion molecules ICAM-1 and LFA-3 can interact with LFA-1 and CD2, respectively, on the T cell. It has been reported that naive T cells may have more stringent requirements for allo-immune activation than memory T cells. This may not only involve the expression of costimulatory and adhesion molecules but also the expression of HLA class II on the APC. The absence of one or more of these molecules on the APC may result in activation of memory T cells but ineffective activation of naive T cells [17–22]. Because UCB contains primarily naive T cells whereas AB contains both naive and memory T cells, T cell populations from these sources are likely to have different requirements for T cell activation with regard to the expression of HLA class II and costimulatory and adhesion molecules on the APC. Another explanation for the reduced alloreactivity in UCB T cells may be a relative immaturity of naive UCB T cells compared with naive AB T cells. Several studies described a lower magnitude of activation of naive UCB T cells as compared with naive T cells from AB in response to CD2 triggering or allogeneic activation [8, 23, 24]. This suggests that naive UCB T cells may have activation requirements that are different from naive AB T cells. In this study, we investigated whether UCB T cells and AB T cells were differentially activated by APC with similar expression of costimulatory and adhesion molecules but with or without the expression of HLA class II
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and whether potential differences were caused by the presence of memory T cells in AB but not in UCB, or also by intrinsic differences between naive T cells from UCB and AB. The responses to allogeneic stimulator cells of naive UCB T cells were compared with naive CD45RA⫹ T cells and memory CD45RO⫹ T cells isolated from AB. As allogeneic stimulator APC, we used the cell line AML193 expressing the costimulatory molecules and adhesion molecules CD86, CD40, ICAM-1, and LFA-3, and HLA class I, but not HLA class II, as well as AML193 cells induced by IFN-␥ to also express HLA-DR. Quantification of interleukin (IL)–2, IL-4, IL-10 or IFN-␥–secreting T cells by enzyme-linked immunospot (ELISPOT) analysis was used to measure T cell activation on days 1–3 after stimulation with the different APC. By measuring the early induction phase of the immune response on days 1–3 after stimulation instead of the measuring proliferation by [3H] thymidine incorporation when extensive cell division is present, the likelihood of bystander activation during culture can be reduced. We demonstrate that naive UCB T cells but not naive AB T cells required the expression of HLA class II on the APC for effective allo-immune activation, resulting in a preferential type 1 response with T cells secreting IL-2 or IFN-␥ but almost no IL-4 or IL-10. Moreover, T cell activation was induced with slower kinetics and at a lower level in naive UCB T cells as compared with naive AB T cells. MATERIALS AND METHODS Collection and Preparation of Peripheral Blood and UCB Samples After informed consent was obtained, peripheral blood cells were obtained from healthy adult donors (n⫽6). UCB cells were derived from the umbilical vein after normal full-term deliveries (n⫽5). Peripheral blood mononuclear cells (PBMC) and UCB mononuclear cells were isolated by centrifugation over a Ficoll-Hypaque gradient and cryopreserved for future use. AML193 Cell Line and Culture Conditions The granulocyte-macrophage colony-stimulating factor (GM-CSF)– dependent human acute myeloid leukemic (AML) cell line AML193 was obtained from the ATCC (Rockville, MD). The AML193 cells were cultured in IMDM (BioWhittaker, Verviers, Belgium) containing 10% human serum supplemented with 20 ng/ml rhGMCSF (Novartis, Bern, Switzerland) in the presence or absence of 1000 IU/ml IFN-␥ (Strathmann Biotech, Hannover, Germany). Before use as stimulator cells, GM-CSF and IFN-␥ were completely removed by washing the AML193 cells three times with IMDM.
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Flow Cytometric Analysis AML193 cells were stained with fluorescein isothiocyanate (FITC)– conjugated anti–HLA-ABC (Becton Dickinson [BD], San Jose, CA), anti-CD40 (Serotec, Kidlington, United Kingdom), anti-CD86 (Pharmingen, San Diego, CA), anti–ICAM-1 (CLB, Amsterdam, The Netherlands) or anti–LFA-3 (Southern Biotechnology Associates, Birmingham, AL) antibodies, or phycoerythrin (PE)–conjugated anti–HLA-DR (BD), or antiCD80 (BD) antibodies. Purity of isolated T cell populations and CD45RA⫹ or CD45RO⫹ T cell fractions was analyzed by labeling with APC-conjugated antiCD3 (BD) antibody, or FITC or PE-conjugated antiCD45 (BD), anti-CD45RA (Coulter, Miami, FL), antiCD45RO (DAKO, Glostrup, Denmark), anti-CD14 (BD), anti-CD19 (BD) antibodies, or glycophorin A (GPA) (Dako) antibodies. Stimulated T cell cultures were labeled with APC-conjugated anti-CD3 and PEconjugated anti-CD25 (BD) or FITC-conjugated antiCD69 (BD). After labeling for 30 minutes at 4°C, the cell samples were washed and analyzed on a FACScan (BD). Isolation of T Cell Populations The mononuclear cell suspensions were thawed, and T cells were purified from the PBMC and UCB mononuclear cells by negative immunomagnetic bead selection with the pan T cell isolation kit from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany), according to the manufacturer’s procedure. This kit contains a cocktail of CD11b, CD16, CD19, CD36, and CD56 antibodies removing B cells, monocytes, NK cells, DC, early erythroid cells, platelets, and basophils. With this negative isolation procedure, CD8⫹ effector T cells that express CD11b are also removed [25]. AB T cell populations from 4 donors were further separated into CD45RA⫹ or CD45RO⫹ T cells by labeling with PE-conjugated CD45RA monoclonal antibodies, followed by immunomagnetic bead selection with anti-PE microbeads (Miltenyi Biotec) or by direct immunomagnetic bead selection with CD45RO microbeads (Miltenyi Biotec). In the purified T cell populations, ⬎95% of the white blood cells were CD3⫹. The purified T cell populations were only slightly contaminated with CD14⫹ monocytes (0.07–%1.9%) and CD19⫹ B cells (0.02%–1.0%). The purified CD45RA⫹ fractions contained 95%–98% CD45RA⫹ cells and 0%–1.5% contaminating CD45RO⫹ cells, and the purified CD45RO⫹ fractions contained 83%–95% CD45RO⫹ cells and 1.1%–14% contaminating CD45RA⫹ cells. Allogeneic T Cell Stimulation The different T cell fractions were resuspended in IMDM containing 10% human serum and were seeded into
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wells of 24-well plates at a concentration of 5 ⫻ 105 T cells/well. The T cell fractions were cultured without stimulator cells, or cocultured with 25-Gy irradiated AML193 cells at a responder:stimulator ratio of 2:1 at a final cell concentration of 7.5 ⫻ 105 cells/well in 1 ml IMDM containing 10% human serum. The cells were cultured at 37°C, 5% CO2, and 95% humidity. On days 1– 4 after stimulation, the supernatant was harvested, and the cells were collected, washed, resuspended in IMDM containing 10% human serum, and the numbers of viable T cells determined. The presence of irradiated stimulator cells did not interfere with this enumeration of viable T cells because T cells could be distinguished from the AML193 cells on the basis of cell size. Proliferation Assay Unstimulated or stimulated T cell cultures were harvested on day 3 of culture, resuspended in IMDM containing 10% human serum, and seeded at concentrations of 5 ⫻ 104 cells per well in 96-well U-bottom microtiter plates in a final volume of 100 l/well. To each well, 1 Ci [3H]thymidine (Amersham, Buckinghamshire, UK) was added. After incubation for an additional 6 hours, the cells were harvested, and [3H]thymidine incorporation was measured by a Topcount NXT (Canberra Packard, Meriden, Connecticut). Data are presented as stimulation indices calculated by cpm of stimulated T cell cultures divided by cpm of unstimulated T cell cultures. ELISPOT Analysis ELISPOT assays for IL-2, IL-4, IL-10 or IFN-␥ were performed with the ELISPOT kits from U-CyTech (Utrecht, The Netherlands) according to the manufacturer’s procedure. Briefly, ELISPOT plates were coated overnight at 4°C with the coating antibodies diluted in phosphate-buffered saline (PBS). After incubation, the wells were washed 5 times with PBS containing 0.05% Tween-20 (washing buffer) and incubated for 1 hour at 37°C with 200 l 1% BSA in PBS. The postcoating solution was removed, and the cell suspensions were added to the wells at concentrations of 5 ⫻ 104, 2.5 ⫻ 104, 1.25 ⫻ 104, and 0.5 ⫻ 104 T cells/well in a final volume of 100 l IMDM containing 10% human serum. After incubation for 5 hours at 37°C, 5% CO2, and 95% humidity, cell suspensions were removed from the wells, and 200 l of sterile water was added to each well. The plates were incubated for 10 minutes on ice and washed 10 times with washing buffer, and then the wells were incubated with 100 l of diluted biotinylated detector antibody solution for 1 hour at 37°C. The wells were washed five times with washing buffer, 50 l of labeled antibiotin antibody was added, and wells were incubated overnight at 4°C. The wells were washed five times with washing buffer, and 30 l of activator solu-
Cord Blood T Cells Need HLA-DR for Allo-Activation
FIGURE 1 Expression of HLA class I, HLA class II, and costimulatory and adhesion molecules on AML193 cells. AML193 cells were cultured in the absence of IFN-␥ (top; AML193-DR⫺) or in the presence of IFN-␥ (bottom; AML193-DR⫹).
tion was added to each well. After incubation for 15 minutes to allow spot formation, the reaction was stopped by rinsing the wells with distilled water. The number of spots was counted on a computer-assisted ELISPOT image analyzer (Olympus Optical, Hamburg, Germany). Statistical Analysis Differences in the numbers of cytokine-secreting T cells between UCB T cells and AB T cells, and between UCB T cells and subpopulations of AB T cells were tested by the Mann-Whitney U test. RESULTS Expression of HLA Class I, HLA Class II, and Costimulatory and Adhesion Molecules on Stimulator Cells As allogeneic stimulator APC, we used the cell line AML193 cultured in the presence of GM-CSF with or without the addition of IFN-␥. The expression of HLA class I, HLA class II, and costimulatory and adhesion molecules on the AML193 cells cultured under these different conditions, as was analyzed by flow cytometry, is illustrated in Figure 1. The AML193 cells cultured without IFN-␥ expressed HLA class I, costimulatory molecules CD40 and CD86, and adhesion molecules CD54 and CD58, but did not express HLA class II or the costimulatory molecule CD80. This cell line will be referred to as AML193-DR⫺. When cultured in the presence of IFN-␥, the AML193 cells additionally expressed HLA-DR. The expression levels of HLA class I, CD80, CD86, CD40, CD54, and CD58 were similar to the expression levels on the AML193 cells cultured in the
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absence of IFN-␥. This cell line will be referred to as AML193-DR⫹. Proliferation After Allogeneic Stimulation Analysis of proliferation by [3H] thymidine incorporation after several days of in vitro culture is the most frequently used method for the analysis of alloreactivity. To analyze whether this assay could also be used for analysis of the early induction phase of the immune response, UCB T cells (n⫽3) or AB T cells (n⫽4) were cocultured with irradiated AML193-DR⫹ cells or AML193-DR⫺ cells, and [3H] thymidine incorporation was measured on day 3. As listed in Table 1, the stimulation indices were low both for UCB T cells (0.8 –1.7) and for AB T cells (0.8 –2.3) when stimulated with AML193-DR⫹ cells or AML193-DR⫺ cells, indicating that [3H] thymidine incorporation was not sensitive enough to measure the limited proliferative response of precursor T cells on day 3 after stimulation. Expression of T Cell Activation Markers CD25 or CD69 After Allogeneic Stimulation Next, we analyzed whether the T cell activation induced by stimulation with the allogeneic APC could be accurately measured by flow cytometric analysis of CD25 or TABLE 1 Stimulation index for UCB T cells or AB T cells on day 3 after stimulation with AML193-DR⫺ cells or AML193-DR⫹ cells Stimulation index Responder
Donor
AML193-DR⫺
AML193-DR⫹
UCB T cells
1 2 3 1 2 3 4
0.8 1 1.4 1.9 1.1 1.7 0.9
0.9 1.2 1.7 0.8 1.2 2.3 1.3
AB T cells
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FIGURE 2 Expression of CD25 or CD69 in unstimulated or stimulated UCB T cells and AB T cells. The percentages of T cells expressing CD25 (A) or CD69 (B) in UCB T cell populations (open symbols; n⫽2 for CD25 and n⫽3 for CD69) and in AB T cell populations (solid symbols; n⫽3 for CD25 and n⫽4 for CD69) after culture without stimulation or after culture with AML193-DR⫹ cells or AML193-DR⫺ cells was measured by flow cytometry on days 1–3 of culture.
CD69 expression on UCB T cells and AB T cells on days 1–3 after stimulation. Figure 2 illustrates the percentages of CD25 or CD69 expressing T cells in UCB T cells or AB T cells when cultured with or without the different stimulator APC. The levels of CD25 expressing T cells in the unstimulated UCB T cells or AB T cells were relatively high on days 1–3 of culture. After stimulation with the different APC no clear increase in the percentages of CD25 expressing T cells in UCB T cells or AB T cells could be detected (Figure 2A). The percentages of CD69 expressing T cells in the unstimulated UCB T cells were low whereas in a number of unstimulated AB T cell fractions relatively high and variable percentages of CD69 expressing T cells were present. The limited changes in percentages of CD69 expressing T cells found for the individual UCB T cell donors or AB T cell donors when stimulated with AML193-DR⫹ cells or AML193-DR⫺ cells were at the detection limit of the flow cytometric method and were highly variable, indicating that the analysis of CD25 or CD69 expressing T cells was not suitable for analyzing the early induction of T cell acti-
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vation on days 1–3 after stimulation with allogeneic APC. Presence of Cytokine-Secreting Cells in UCB T Cell and AB T Cell Populations After Stimulation With AML193-DRⴙ Cells To determine whether allogeneic stimulation under the most optimal circumstances, by optimal APC expressing HLA class I, HLA class II, and costimulatory and adhesion molecules resulted in differential cytokine secretion by naive UCB T cells or AB T cells, the numbers of IL-2–, IFN-␥–, IL-4 –, or IL-10 –secreting T cells on days 1–3 after stimulation with AML193-DR⫹ cells were measured by ELISPOT analysis. The AML193DR⫹ cells did not produce any of the cytokines studied (data not shown). In the unstimulated T cell fractions from UCB, no cytokine-secreting cells were present. The unstimulated AB T cell fractions contained no IL-4 – secreting cells, whereas the numbers of T cells spontaneously secreting IL-2, IFN-␥, or IL-10 ranged from 0 – 4, 0 –27, and 0 –22 per 50,000 cells, respectively. The numbers of cytokine-secreting T cells after stimulation with the AML193-DR⫹ cells were corrected for the numbers of cytokine-secreting cells in the unstimulated T cell populations. AML193-DR⫹ cells did not induce IL-4 secretion in UCB T cells or in AB T cell fractions (0 –3 IL-4 –secreting cells per 50,000 viable T cells). No IL-10 secretion was induced in UCB T cells or AB T cells on days 1 and 2 after stimulation with AML193-DR⫹ cells (data not shown). Three days after stimulation,
Cord Blood T Cells Need HLA-DR for Allo-Activation
FIGURE 3 Numbers of IL-2– or IFN-␥–secreting cells in UCB T cells and AB T cells on days 1–3 after stimulation with AML193-DR⫹ cells. Purified UCB T cells (open symbols; n⫽5) and AB T cells (solid symbols; n⫽5) were stimulated with AML193-DR⫹ cells. On days 1–3 the numbers of IL-2– (A) or IFN-␥– (B) secreting cells per 50,000 viable T cells were determined by ELISPOT analysis. Bars represent medians. Numbers are corrected for the unstimulated T cell populations. *Significant difference between UCB T cells and AB T cells (p ⬍ 0.05).
IL-10 –secreting cells were present at comparable low levels in both UCB T cells and AB T cells ranging from 2–15 and 0 –26 per 50,000 viable UCB T cells and AB T cells, respectively. The numbers of IL-2– or IFN-␥–secreting T cells on days 1–3 after stimulation with AML193-DR⫹ cells are illustrated in Figure 3. AML193-DR⫹ cells induced IL-2 secretion on day 1 in the UCB T cell populations, which did not change on days 2 and 3 after stimulation. In AB T cells, AML193-DR⫹ cells induced IL-2 secretion on day 1, which increased in some cases on days 2 and 3, although the numbers of IL-2–secreting cells between UCB T cells and AB T cells were not statistically different because of the large variation (Figure 3A). AMLDR⫹ cells did not induce IFN-␥ secretion in UCB T cells on day 1 and 2 after stimulation, and on day 3, only limited numbers of IFN-␥–secreting cells were induced. In contrast, AML193-DR⫹ cells induced IFN-␥–secreting cells in AB T cells on days 1–3 after stimulation (Figure 3B).
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FIGURE 4 Numbers of IL-2– or IFN-␥–secreting cells in UCB T cells, and CD45RA⫹ or CD45RO⫹ T cell fractions from AB on days 1–3 after stimulation with AML193-DR⫹ cells. UCB T cells, and CD45RA⫹ or CD45RO⫹ T cell fractions from AB were stimulated with AML193-DR⫹ cells, and the numbers of cells secreting IL-2 (A) or IFN-␥ (B) per 50,000 viable T cells were determined by ELISPOT analysis on days 1–3 after stimulation. Each symbol represents a single donor with open circles for UCB donors (n⫽5), solid circles for CD45RA⫹ T cells from AB (n⫽4), and solid triangles for CD45RO⫹ T cells from AB (n⫽4). Bars represent medians. Numbers are corrected for unstimulated T cell populations. *Significant difference between UCB T cells and CD45RA⫹ T cells from AB; indicates a significant difference between UCB T cells and CD45RO⫹ T cells from AB (p ⬍ 0.05). No statistically significant differences between CD45RA⫹ T cells and CD45RO⫹ T cells from AB were present.
IL-2– or IFN-␥–Secreting Cells in Naive CD45RAⴙ or Memory CD45ROⴙ T Cell Fractions From AB After Stimulation With AML-DRⴙ Cells To analyze whether differences in IFN-␥ or IL-2 secretion between UCB T cells and AB T cells in response to AML193-DR⫹ cells were caused by the presence of memory T cells in AB, or whether there was also an intrinsic difference between the naive CD45RA⫹ T cells from UCB and AB, naive and memory T cell fractions were separated from the AB T cells on the basis of CD45RA or CD45RO expression. In Figure 4, the numbers of IL-2– or IFN-␥–secreting cells present in UCB T cells are compared with the numbers of IL-2– or IFN␥–secreting cells measured in the CD45RA⫹ or the CD45 RO⫹ T cell fractions from AB at days 1–3 after
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FIGURE 5 Numbers of IL-2– or IFN-␥–secreting cells in UCB T cells and AB T cells after stimulation with AML193DR⫹ cells or AML193-DR⫺ cells. On days 1–3 after stimulation with AML193-DR⫹ cells (solid symbols) or AML193DR⫺ cells (open symbols), the numbers of IL-2– (A and C) or IFN-␥– (B and D) secreting cells per 50,000 viable T cells were determined in UCB T cells (n⫽5 for stimulation with AML193-DR⫹ cells and n⫽4 for stimulation with AML193DR⫺ cells) and in AB T cells (n⫽5). Values are corrected for unstimulated controls. *Significant difference between stimulation with AML193-DR⫹ cells and stimulation with AML193-DR⫺ cells.
stimulation with AML193-DR⫹ cells. Values are corrected for the numbers of cytokine-secreting T cells in the unstimulated T cell populations that ranged from 0 –3 and 0 –11 IL-2–secreting cells and 0 –25 and 0 – 66 IFN-␥–secreting cells per 50,000 cells in the CD45RA⫹ and CD45RO⫹ T cell fractions, respectively. In the unstimulated UCB T cells, no IL-2 or IFN-␥ secretion was present. The numbers of IL-2–secreting cells induced by the AML193-DR⫹ cells in naive UCB T cells or naive CD45RA⫹ T cells from AB were similar and levels did not significantly differ on days 1–3 after stimulation. The numbers of IL-2–secreting cells induced by the AML193-DR⫹ cells in the CD45RO⫹ T cell fraction were also low on day 1 but increased significantly on days 2 and 3 after stimulation as compared with UCB T cells (Figure 4A). In contrast, UCB T cells responded to AML193-DR⫹ with only low numbers of IFN-␥–secreting cells on day 3 after stimulation. High numbers of IFN-␥–secreting cells were induced in both the CD45RA⫹ and the
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CD45RO⫹ T cell fractions from AB on days 1 and 2 after stimulation, which was significantly different from UCB T cells (Figure 4B). In conclusion, the difference in IL-2 secretion between UCB T cells and AB T cells in response to stimulation with AML193-DR⫹ cells appeared to be caused by the presence of memory T cells in AB, whereas the difference in IFN-␥ secretion in response to stimulation with AML193-DR⫹ cells was due to a difference in IFN-␥ secretion between naive UCB T cells and both CD45RA⫹ naive T cells and CD45RO⫹ memory T cells from AB. Cytokine-Secreting Cells in T Cell Populations From UCB or AB After Stimulation With AML193-DRⴚ Cells To determine whether the expression of HLA class II on the APC was essential for the induction of cytokine secretion in UCB T cells or AB T cells, the numbers of IL-2–, IFN-␥–, IL-4 –, or IL-10 –secreting cells in UCB T cells or AB T cells on days 1–3 after stimulation with AML193-DR⫹ cells or AML193-DR⫺ cells were compared. Similar to stimulation with AML193-DR⫹ cells, AML193-DR⫺ cells did not induce IL-4 secretion in UCB T cells or AB T cells (data not shown). IL-10 – secreting cells were present on day 3 in both UCB T cells and AB T cells, at comparable low levels ranging from 1–9 and 0 –25 IL-10 –secreting cells per 50,000 UCB T cells and AB T cells, respectively. Figure 5 illustrates the numbers of IL-2– or IFN-␥– secreting cells in UCB T cells or AB T cells after stimulation with the AML193-DR⫺ cells or AML193-DR⫹
Cord Blood T Cells Need HLA-DR for Allo-Activation
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tions were separated from the AB T cells and the frequencies of IL-2– or IFN-␥–secreting T cells were analyzed after stimulation with AML193-DR⫺ cells. The numbers were corrected for the frequencies of cytokinesecreting cells in the unstimulated T cell populations that ranged from 0 –3 and 0 –11 IL-2–secreting cells, and 0 –25 and 0 – 66 IFN-␥–secreting cells per 50,000 CD45RA⫹ T cells and CD45RO⫹ T cells, respectively. As illustrated in Figure 6 , a significant difference in the numbers of IL-2– or IFN-␥–secreting cells in response to AML193-DR⫺ cells between the CD45RA⫹ naive T cells and the CD45RO⫹ memory T cells was only present for the IL-2 secretion on day 1. These results indicate that AML193-DR⫺ cells induced IL-2– or IFN␥–secreting cells in both CD45RA⫹ naive T cells and CD45RO⫹ memory T cells from AB but not in naive UCB T cells.
FIGURE 6 Numbers of IL-2– or IFN-␥–secreting cells in CD45RA⫹ or CD45RO⫹ T cell fractions from AB on days 1–3 after stimulation with AML193-DR⫺ cells. The CD45RA⫹ (open triangles; n⫽4) or CD45RO⫹ (solid triangles; n⫽4) T cell fractions from AB were stimulated with AML193-DR⫺ cells, and the numbers of cells secreting IL-2 (A) or IFN-␥ (B) per 50,000 viable T cells were determined by ELISPOT analysis on days 1–3 after stimulation. Bars represent medians. Numbers are corrected for unstimulated T cell populations. *Significant difference between CD45RA⫹ T cell fractions and CD45RO⫹ T cell fractions.
cells, corrected for the numbers of cytokine-secreting cells present in the unstimulated T cell populations. The background levels in unstimulated AB T cells ranged from 0 – 4 and 0 –27 cytokine-secreting cells per 50,000 cells for IL-2 and IFN-␥, respectively. In UCB T cells, IL-2–secreting cells were induced by AML193-DR⫹ cells but not by AML193-DR⫺ cells. Low numbers of IFN-␥–secreting cells were induced in the UCB T cells by AML193-DR⫹ cells on day 3 of stimulation, but not by the AML193-DR⫺ cells (p ⬍ 0.05) (Figure 5A,B). In contrast, IL-2– or IFN-␥–secreting cells were induced in AB T cells by stimulation with both the AML193-DR⫹ cells and the AML193-DR⫺ cells (Figure 5C,D). In conclusion, UCB T cells but not AB T cells required the expression of HLA class II for the induction of IL-2 secretion and IFN-␥ secretion. To determine whether, similar to naive UCB T cells, IL-2 secretion and IFN-␥ secretion cannot be induced in CD45RA⫹ naive AB T cells by stimulation with AML193-DR⫺ cells, the naive and memory T cell frac-
DISCUSSION The immunological mechanism responsible for the lower incidence and severity of GVHD found for UCB transplantation as compared with adult SCT has not yet been elucidated. In several comparative studies measuring the proliferative, cytotoxic, and cytokine responses of UCB T cells and AB T cells after allogeneic stimulation, a lower alloreactivity was found for UCB T cells [4 –10]. To further investigate the underlying conditions for a reduced alloreactivity in UCB T cells, we analyzed whether UCB T cells and AB T cells have differential requirements for the presence of HLA class II on the APC for the induction of T cell activation, and whether differences were only caused by the presence of memory T cells in AB or also by intrinsic differences between naive T cells from UCB and AB. Because PBMC or cultured DC used as allogeneic stimulator cells are heterogeneous and have variable expressions of HLA class I, HLA class II, and costimulatory and adhesion molecules, we used a cell line with defined and fixed levels of these surface molecules as allogeneic stimulator cells. AML193 cells expressed HLA class I, costimulatory molecules CD86 and CD40 and adhesion molecules ICAM-1 and LFA-3. In addition to the HLA class II–negative AML193 cell line, we used AML193 cells that were induced by IFN-␥ to also express HLADR. The AML193 cell line is derived from a malignant cell and may therefore not reflect all the different APC that are encountered in vivo. Nevertheless, this cell line expressed most of the costimulatory and adhesion molecules that are expressed on maturated APC, and that are important for the generation of an immune response. Therefore, the AML193 cell line provides an adequate model system to study the activation requirements of UCB T cells and AB T cells.
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To analyze the early induction phase of the immune response, we attempted to measure T cell activation on days 1–3 after stimulation of purified T cells from AB or UCB with the different APC. Differences in alloreactivity are often studied by analysis of proliferation by [3H] thymidine incorporation after 5 or more days of in vitro culture [4, 6, 26, 27]. Because 2–3 days after stimulation activated T cells start to rapidly proliferate, analysis later than 3 days of culture will strongly be influenced by the proliferation rate of the responding T cells. However, our results indicate that as early as day 3, [3H] thymidine incorporation was not sensitive enough to accurately measure proliferation of the limited numbers of activated precursor T cells. The analysis of CD25 or CD69 expression by flow cytometry was also found to be not sensitive enough to measure the induction of T cell activation on days 1–3 after stimulation with the different APC. This was in part because of high and variable percentages of CD25 or CD69 expressing T cells present in the unstimulated T cell cultures. Relatively high percentages of CD25 expressing regulatory T cells in unstimulated T cell cultures may be responsible for these findings [28, 29]. The analysis of cytokine-secreting T cells by ELISPOT analysis was found to be sensitive enough to detect T cell activation after 1–3 days of stimulation and was therefore used to further analyze the requirements for the induction of T cell activation in UCB T cells and AB T cells. The analysis of cytokine-secreting T cells was not performed at a later stage than 3 days after stimulation because beyond that time point, rapid cell division is likely to occur. Cell division can strongly influence cytokine secretion, especially during the S-phase of the cell cycle until completion of cell division. Accurate analysis of cytokine secretion at the single cell level can therefore only be performed on days 1–3 after stimulation when extensive cell division is not yet apparent. Because not only the number of activated T cells but also the balance between secreted type 1 cytokines and type 2 cytokines may be important for the clinical outcome of allogeneic responses [30], the numbers of T cells secreting the type 1 cytokines IL-2 or IFN-␥ as well as the numbers of T cells secreting the type 2 cytokines IL-4 or IL-10 were measured. Stimulation with AML193-DR⫹ cells or AML193DR⫺ cells induced a preferential type 1 cytokine response in both UCB T cells and AB T cells with IL-2– or IFN-␥–secreting cells, but almost no IL-4 – or IL-10 – secreting cells. These data are in line with other studies describing the presence of IL-4 – or IL-10 –secreting T cells only after repetitive stimulation with PBMC or DC and not after primary stimulation [4, 8, 31–35] and suggest that IL-4 and IL-10 may only be produced after
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T cell differentiation and not at the initial induction phase of the allo-immune response. Under the most optimal conditions of T cell activation in the presence of both HLA class I and class II on the AML193 cells, IL-2– or IFN-␥–secreting T cells were induced in both UCB T cells and AB T cells. However, the induction of IL-2 secretion and IFN-␥ secretion was slower in UCB T cells, and the numbers of IL-2– or IFN-␥–secreting cells in UCB T cells were lower as compared with AB T cells, indicating a slower kinetics and a lower level of activation in UCB T cells. These data are consistent with studies that describe less secretion of IL-2 or IFN-␥ for UCB T cells as compared with AB T cells after allogeneic stimulation [4, 36]. The differences in kinetics and levels of activation between UCB T cells and AB T cells were not only due to the presence of memory T cells in AB T cells but also due to an intrinsic difference between naive T cells from UCB and from AB. Stimulation of naive CD45RA⫹ T cell fractions and memory CD45RO⫹ T cell fractions with the AML193-DR⫹ cells exhibited an earlier induction and a higher level of IL-2 secretion for the CD45RO⫹ memory T cells but not for the CD45RA⫹ naive T cells from AB, as compared with UCB T cells. In contrast, IFN-␥ secretion was induced earlier and reached a higher level as compared with UCB T cells in both the memory CD45RO⫹ T cells and the naive CD45RA⫹ T cells from AB. For these comparisons with naive UCB T cells, the high purity of the isolated CD45RA⫹ T cell fractions was crucial. The contaminating naive CD45RA⫹ T cells in the memory CD45RO⫹ T cell fractions were found not to significantly influence the outcome as the response induced in memory CD45RO⫹ T cells was higher or comparable to the response induced in naive CD45RA⫹ T cells. On day 1 after stimulation with the AML193-DR⫹ cells, the numbers of IL-2–secreting T cells in the naive CD45RA⫹ T cells and memory CD45RO⫹ T cells were much lower than the numbers of numbers of IFN-␥– secreting T cells in these T cell fractions. This difference is probably due to the presence of large numbers of single T cells that do not produce both cytokines simultaneously but instead produce only one of the two cytokines, with most single T cells secreting only IFN-␥. The presence of such single IL-2– or IFN-␥–secreting T cells in response to different stimuli has also been reported by others [37, 38], and is in agreement with the notion that the separation of T cells into type 1, type 2 and type 0 cytokine-secreting T cells is less strict in humans as compared with mice. The decline in IFN-␥–secreting cells in these two T cell fractions on day 3 after stimulation is most likely caused by the kinetic fashion in which cytokines are produced by single cells [37, 39, 40].
Cord Blood T Cells Need HLA-DR for Allo-Activation
Not only the kinetics and level of activation but also the requirement for HLA-DR expression on the APC for the induction of activation was found to be different for UCB T cells and AB T cells. The expression of HLA-DR on the AML193 cells was found to be essential for the activation of UCB T cells but not for AB T cells, as demonstrated by the virtual absence of IL-2– or IFN-␥– secreting T cells in UCB T cells when stimulated with the AML193-DR⫺ cells whereas the numbers of IL-2– or IFN-␥–secreting T cells present in AB T cells after stimulation with AML193-DR⫺ cells were comparable to the numbers after stimulation with the AML193DR⫹ cells. Because AML193-DR⫺ cells cannot activate CD4⫹ T cells the responses measured after stimulation with the AML193-DR⫺ cells are likely to be derived from the CD8⫹ T cells. Apparently, CD8⫹ T cells from AB but not the CD8⫹ T cells from UCB could respond to stimulation with the AML193-DR⫺ cells. Whether naive CD8⫹ T cells from UCB required the help of CD4⫹ T cells for their activation or whether naive UCB CD8⫹ T cells were completely irresponsive and did also not respond to stimulation with the AML193-DR⫹ cells could not be determined because we did not study CD4⫹ cells and CD8⫹ T cells separately. The capability to respond to APC that did not express HLA-DR was not only found for the memory CD45RO⫹ T cells but also for the naive CD45RA⫹ T cells from AB as both AB T cell fractions secreted IL-2 and IFN-␥ in response to stimulation with the AML-DR⫺ cells. These data further indicated that naive UCB T cells and naive T cells from AB are intrinsically different, with naive UCB T cells but not naive CD45RA⫹ from AB requiring the presence of HLA class II on the APC for allogeneic activation. Furthermore, the observations that the AML193-DR⫹ cells but not the AML193-DR⫺ cells induced cytokine-secreting cells in UCB T cells illustrate that it is highly unlikely that the effects measured in this study are caused by nonspecific stimulation by the AML193 cells. The differences in allo-immune activation between UCB T cells and AB T cells that we found in this study may help to explain the lower GVHD found after UCB transplantation. Naive UCB T cells but not naive or memory T cells from AB may be unresponsive to vascular endothelial cells that under normal circumstances do not express HLA-DR [41] and that are the first APC to be encountered by the donor T cells after transplantation. Even when UCB T cells will be activated by the encounter of APC that express HLA-DR, such as endothelial cells, that have upregulated HLA class II as a result of in vivo inflammatory responses or professional APC like DC, cytokine secretion may be induced more slow in UCB T cells and the level of the cytokine response may be lower as compared with that induced in naive or
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memory T cells from AB, possibly resulting in less severe GVHD. In conclusion, naive UCB T cells but not AB naive T cells require the expression of HLA-DR on the APC for allo-immune activation. Furthermore, in naive UCB T cells allo-immune activation was induced with slower kinetics and at a lower level, as compared with AB naive T cells. These findings may in part explain the reduced incidence and severity of GVHD after UCB transplantation. REFERENCES 1. Rocha V, Cornish J, Sievers EL, Filipovich A, Locatelli F, Peters C, Remberger M, Michel G, Arcese W, Dallorso S, Tiedemann K, Busca A, Chan KW, Kato S, Ortega J, Vowels M, Zander A, Souillet G, Oakill A, Woolfrey A, Pay AL, Green A, Garnier F, Ionescu I, Wernet P, Sirchia G, Rubinstein P, Chevret S, Gluckman E: Comparison of outcomes of unrelated bone marrow and umbilical cord blood transplants in children with acute leukemia. Blood 97:2962, 2001. 2. Rocha V, Wagner Jr JE, Sobocinski KA, Klein JP, Zhang MJ, Horowitz MM, Gluckman E: Graft-versus-host disease in children who have received a cord-blood or bone marrow transplant from an HLA-identical sibling. Eurocord and International Bone Marrow Transplant Registry Working Committee on Alternative Donor and Stem Cell Sources. N Engl J Med 342:1846, 2000. 3. Grewal SS, Barker JN, Davies SM, Wagner JE: Unrelated donor hematopoietic cell transplantation: marrow or umbilical cord blood? Blood 101:4233, 2003. 4. Risdon G, Gaddy J, Stehman FB, Broxmeyer HE: Proliferative and cytotoxic responses of human cord blood T lymphocytes following allogeneic stimulation. Cell Immunol 154:14, 1994. 5. Harris DT, LoCascio J, Besencon FJ: Analysis of the alloreactive capacity of human umbilical cord blood: implications for graft-versus-host disease. Bone Marrow Transplant 14:545, 1994. 6. Roncarolo MG, Bigler M, Ciuti E, Martino S, Tovo PA: Immune responses by cord blood cells. Blood Cells 20: 573, 1994. 7. Keever CA, Abu-Hajir M, Graf W, McFadden P, Prichard P, O’Brien J, Flomenberg N: Characterization of the alloreactivity and anti-leukemia reactivity of cord blood mononuclear cells. Bone Marrow Transplant 15:407, 1995. 8. Slavcev A, Strı¨z I, Ivasˇkova´ E, Breur-Vriesendorp BS: Alloresponses of cord blood cells in primary mixed lymphocyte cultures. Hum Immunol 63:155, 2002. 9. Harris DT, Schumacher MJ, LoCascio J, Besencon FJ, Olson GB, DeLuca D, Shenker L, Bard J, Boyse EA: Phenotypic and functional immaturity of human umbili-
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INTRODUCTION Allogeneic stem cell transplantation (SCT) has been successfully used for the treatment of various hematologic and nonhematologic diseases. One of the major complications that can occur after allogeneic SCT that uses stem cell grafts from adult donors is graft-versus-host disease
From the Department of Hematology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. Address reprint requests to: Dr. F.M. Kloosterboer, Department of Hematology, Leiden University Medical Center, C2-R, P.O. Box 9600, 2300 RC Leiden, The Netherlands; Tel: ⫹31 71 526 2271; Fax ⫹31 71 526 6755; E-mail: [email protected]. Received October 6, 2003; revised December 30, 2003; accepted January 5, 2004. Human Immunology 65, 328 –339 (2004) © American Society for Histocompatibility and Immunogenetics, 2004 Published by Elsevier Inc.
IL-2 but higher IFN-␥ secretion in CD45RA⫹ T cells from AB than in UCB T cells. AML193-DR⫺ cells did not induce IL-2– or IFN-␥ secretion in UCB T cells, but stimulated both CD45RA⫹ and CD45RO⫹ T cells from AB to secrete IL-2 and IFN-␥. Thus, not only the absence of memory T cells but also the inability to respond to HLA-DR–negative antigen-presenting cells and the slower kinetics and level of activation found for naive T cells from UCB as compared with AB may partly explain the reduced antirecipient reactivity after UCB transplantation. Human Immunology 65, 328 –339 (2004). © American Society for Histocompatibility and Immunogenetics, 2004. Published by Elsevier Inc. KEYWORDS: umbilical cord blood; graft-versus-host disease; alloreactivity; HLA class II; T cells
IFN-␥ IL PBMC SCT UCB
interferon-gamma interleukin peripheral blood mononuclear cells stem cell transplantation umbilical cord blood
(GVHD). In contrast, when umbilical cord blood (UCB) is used as stem cell source for SCT, a relatively low incidence and severity of GVHD has been found in both human leukocyte antigen (HLA) identical and partially matched unrelated UCB transplants [1–3]. A diminished alloreactivity in UCB T cells has been proposed as a possible mechanism responsible for the lower incidence and severity of GVHD after UCB transplantation. Some investigators have described diminished proliferative or cytotoxic responses of UCB T cells as compared with adult blood (AB) T cells in mixed lymphocyte reaction or in limiting dilution analysis measuring the frequencies of antirecipient helper T precursor 0198-8859/04/$–see front matter doi:10.1016/j.humimm.2004.01.003
Cord Blood T Cells Need HLA-DR for Allo-Activation
cells and cytotoxic T precursor cells [4 –10]. However, others have reported similar proliferative or cytotoxic capacity for UCB T cells and AB T cells in response to allogeneic activation [4, 6, 10 –12]. Allogeneic activation of CD4⫹ T cells and CD8⫹ T cells requires T cell receptor (TCR) signaling mediated through interaction with HLA class I or HLA class II molecules, respectively, on the antigen-presenting cells (APC). Whereas nearly all cells express HLA class I, constitutive expression of HLA class II is mainly limited to professional APC, like dendritic cells (DC), except for expression on hematopoietic progenitor cells. Expression of HLA class II molecules can, however, be induced by proinflammatory cytokines, such as interferon gamma (IFN-␥) or tumor necrosis factor alpha. It has been demonstrated that CD4⫹ helper activity is required for the generation of CD8⫹ T cell responses, and as a consequence the expression of MHC class II on the APC may be essential for the activation of both CD4⫹ T cells and CD8⫹ T cells. Effective T cell activation is further facilitated by additional signaling through costimulatory and adhesion molecules that can be expressed on the APC [13–17]. The most important costimulatory molecules are CD40, which can interact with CD40 ligand on the T cell, and CD80 or CD86, which can interact with CD28 on the T cell. The adhesion molecules ICAM-1 and LFA-3 can interact with LFA-1 and CD2, respectively, on the T cell. It has been reported that naive T cells may have more stringent requirements for allo-immune activation than memory T cells. This may not only involve the expression of costimulatory and adhesion molecules but also the expression of HLA class II on the APC. The absence of one or more of these molecules on the APC may result in activation of memory T cells but ineffective activation of naive T cells [17–22]. Because UCB contains primarily naive T cells whereas AB contains both naive and memory T cells, T cell populations from these sources are likely to have different requirements for T cell activation with regard to the expression of HLA class II and costimulatory and adhesion molecules on the APC. Another explanation for the reduced alloreactivity in UCB T cells may be a relative immaturity of naive UCB T cells compared with naive AB T cells. Several studies described a lower magnitude of activation of naive UCB T cells as compared with naive T cells from AB in response to CD2 triggering or allogeneic activation [8, 23, 24]. This suggests that naive UCB T cells may have activation requirements that are different from naive AB T cells. In this study, we investigated whether UCB T cells and AB T cells were differentially activated by APC with similar expression of costimulatory and adhesion molecules but with or without the expression of HLA class II
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and whether potential differences were caused by the presence of memory T cells in AB but not in UCB, or also by intrinsic differences between naive T cells from UCB and AB. The responses to allogeneic stimulator cells of naive UCB T cells were compared with naive CD45RA⫹ T cells and memory CD45RO⫹ T cells isolated from AB. As allogeneic stimulator APC, we used the cell line AML193 expressing the costimulatory molecules and adhesion molecules CD86, CD40, ICAM-1, and LFA-3, and HLA class I, but not HLA class II, as well as AML193 cells induced by IFN-␥ to also express HLA-DR. Quantification of interleukin (IL)–2, IL-4, IL-10 or IFN-␥–secreting T cells by enzyme-linked immunospot (ELISPOT) analysis was used to measure T cell activation on days 1–3 after stimulation with the different APC. By measuring the early induction phase of the immune response on days 1–3 after stimulation instead of the measuring proliferation by [3H] thymidine incorporation when extensive cell division is present, the likelihood of bystander activation during culture can be reduced. We demonstrate that naive UCB T cells but not naive AB T cells required the expression of HLA class II on the APC for effective allo-immune activation, resulting in a preferential type 1 response with T cells secreting IL-2 or IFN-␥ but almost no IL-4 or IL-10. Moreover, T cell activation was induced with slower kinetics and at a lower level in naive UCB T cells as compared with naive AB T cells. MATERIALS AND METHODS Collection and Preparation of Peripheral Blood and UCB Samples After informed consent was obtained, peripheral blood cells were obtained from healthy adult donors (n⫽6). UCB cells were derived from the umbilical vein after normal full-term deliveries (n⫽5). Peripheral blood mononuclear cells (PBMC) and UCB mononuclear cells were isolated by centrifugation over a Ficoll-Hypaque gradient and cryopreserved for future use. AML193 Cell Line and Culture Conditions The granulocyte-macrophage colony-stimulating factor (GM-CSF)– dependent human acute myeloid leukemic (AML) cell line AML193 was obtained from the ATCC (Rockville, MD). The AML193 cells were cultured in IMDM (BioWhittaker, Verviers, Belgium) containing 10% human serum supplemented with 20 ng/ml rhGMCSF (Novartis, Bern, Switzerland) in the presence or absence of 1000 IU/ml IFN-␥ (Strathmann Biotech, Hannover, Germany). Before use as stimulator cells, GM-CSF and IFN-␥ were completely removed by washing the AML193 cells three times with IMDM.
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Flow Cytometric Analysis AML193 cells were stained with fluorescein isothiocyanate (FITC)– conjugated anti–HLA-ABC (Becton Dickinson [BD], San Jose, CA), anti-CD40 (Serotec, Kidlington, United Kingdom), anti-CD86 (Pharmingen, San Diego, CA), anti–ICAM-1 (CLB, Amsterdam, The Netherlands) or anti–LFA-3 (Southern Biotechnology Associates, Birmingham, AL) antibodies, or phycoerythrin (PE)–conjugated anti–HLA-DR (BD), or antiCD80 (BD) antibodies. Purity of isolated T cell populations and CD45RA⫹ or CD45RO⫹ T cell fractions was analyzed by labeling with APC-conjugated antiCD3 (BD) antibody, or FITC or PE-conjugated antiCD45 (BD), anti-CD45RA (Coulter, Miami, FL), antiCD45RO (DAKO, Glostrup, Denmark), anti-CD14 (BD), anti-CD19 (BD) antibodies, or glycophorin A (GPA) (Dako) antibodies. Stimulated T cell cultures were labeled with APC-conjugated anti-CD3 and PEconjugated anti-CD25 (BD) or FITC-conjugated antiCD69 (BD). After labeling for 30 minutes at 4°C, the cell samples were washed and analyzed on a FACScan (BD). Isolation of T Cell Populations The mononuclear cell suspensions were thawed, and T cells were purified from the PBMC and UCB mononuclear cells by negative immunomagnetic bead selection with the pan T cell isolation kit from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany), according to the manufacturer’s procedure. This kit contains a cocktail of CD11b, CD16, CD19, CD36, and CD56 antibodies removing B cells, monocytes, NK cells, DC, early erythroid cells, platelets, and basophils. With this negative isolation procedure, CD8⫹ effector T cells that express CD11b are also removed [25]. AB T cell populations from 4 donors were further separated into CD45RA⫹ or CD45RO⫹ T cells by labeling with PE-conjugated CD45RA monoclonal antibodies, followed by immunomagnetic bead selection with anti-PE microbeads (Miltenyi Biotec) or by direct immunomagnetic bead selection with CD45RO microbeads (Miltenyi Biotec). In the purified T cell populations, ⬎95% of the white blood cells were CD3⫹. The purified T cell populations were only slightly contaminated with CD14⫹ monocytes (0.07–%1.9%) and CD19⫹ B cells (0.02%–1.0%). The purified CD45RA⫹ fractions contained 95%–98% CD45RA⫹ cells and 0%–1.5% contaminating CD45RO⫹ cells, and the purified CD45RO⫹ fractions contained 83%–95% CD45RO⫹ cells and 1.1%–14% contaminating CD45RA⫹ cells. Allogeneic T Cell Stimulation The different T cell fractions were resuspended in IMDM containing 10% human serum and were seeded into
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wells of 24-well plates at a concentration of 5 ⫻ 105 T cells/well. The T cell fractions were cultured without stimulator cells, or cocultured with 25-Gy irradiated AML193 cells at a responder:stimulator ratio of 2:1 at a final cell concentration of 7.5 ⫻ 105 cells/well in 1 ml IMDM containing 10% human serum. The cells were cultured at 37°C, 5% CO2, and 95% humidity. On days 1– 4 after stimulation, the supernatant was harvested, and the cells were collected, washed, resuspended in IMDM containing 10% human serum, and the numbers of viable T cells determined. The presence of irradiated stimulator cells did not interfere with this enumeration of viable T cells because T cells could be distinguished from the AML193 cells on the basis of cell size. Proliferation Assay Unstimulated or stimulated T cell cultures were harvested on day 3 of culture, resuspended in IMDM containing 10% human serum, and seeded at concentrations of 5 ⫻ 104 cells per well in 96-well U-bottom microtiter plates in a final volume of 100 l/well. To each well, 1 Ci [3H]thymidine (Amersham, Buckinghamshire, UK) was added. After incubation for an additional 6 hours, the cells were harvested, and [3H]thymidine incorporation was measured by a Topcount NXT (Canberra Packard, Meriden, Connecticut). Data are presented as stimulation indices calculated by cpm of stimulated T cell cultures divided by cpm of unstimulated T cell cultures. ELISPOT Analysis ELISPOT assays for IL-2, IL-4, IL-10 or IFN-␥ were performed with the ELISPOT kits from U-CyTech (Utrecht, The Netherlands) according to the manufacturer’s procedure. Briefly, ELISPOT plates were coated overnight at 4°C with the coating antibodies diluted in phosphate-buffered saline (PBS). After incubation, the wells were washed 5 times with PBS containing 0.05% Tween-20 (washing buffer) and incubated for 1 hour at 37°C with 200 l 1% BSA in PBS. The postcoating solution was removed, and the cell suspensions were added to the wells at concentrations of 5 ⫻ 104, 2.5 ⫻ 104, 1.25 ⫻ 104, and 0.5 ⫻ 104 T cells/well in a final volume of 100 l IMDM containing 10% human serum. After incubation for 5 hours at 37°C, 5% CO2, and 95% humidity, cell suspensions were removed from the wells, and 200 l of sterile water was added to each well. The plates were incubated for 10 minutes on ice and washed 10 times with washing buffer, and then the wells were incubated with 100 l of diluted biotinylated detector antibody solution for 1 hour at 37°C. The wells were washed five times with washing buffer, 50 l of labeled antibiotin antibody was added, and wells were incubated overnight at 4°C. The wells were washed five times with washing buffer, and 30 l of activator solu-
Cord Blood T Cells Need HLA-DR for Allo-Activation
FIGURE 1 Expression of HLA class I, HLA class II, and costimulatory and adhesion molecules on AML193 cells. AML193 cells were cultured in the absence of IFN-␥ (top; AML193-DR⫺) or in the presence of IFN-␥ (bottom; AML193-DR⫹).
tion was added to each well. After incubation for 15 minutes to allow spot formation, the reaction was stopped by rinsing the wells with distilled water. The number of spots was counted on a computer-assisted ELISPOT image analyzer (Olympus Optical, Hamburg, Germany). Statistical Analysis Differences in the numbers of cytokine-secreting T cells between UCB T cells and AB T cells, and between UCB T cells and subpopulations of AB T cells were tested by the Mann-Whitney U test. RESULTS Expression of HLA Class I, HLA Class II, and Costimulatory and Adhesion Molecules on Stimulator Cells As allogeneic stimulator APC, we used the cell line AML193 cultured in the presence of GM-CSF with or without the addition of IFN-␥. The expression of HLA class I, HLA class II, and costimulatory and adhesion molecules on the AML193 cells cultured under these different conditions, as was analyzed by flow cytometry, is illustrated in Figure 1. The AML193 cells cultured without IFN-␥ expressed HLA class I, costimulatory molecules CD40 and CD86, and adhesion molecules CD54 and CD58, but did not express HLA class II or the costimulatory molecule CD80. This cell line will be referred to as AML193-DR⫺. When cultured in the presence of IFN-␥, the AML193 cells additionally expressed HLA-DR. The expression levels of HLA class I, CD80, CD86, CD40, CD54, and CD58 were similar to the expression levels on the AML193 cells cultured in the
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absence of IFN-␥. This cell line will be referred to as AML193-DR⫹. Proliferation After Allogeneic Stimulation Analysis of proliferation by [3H] thymidine incorporation after several days of in vitro culture is the most frequently used method for the analysis of alloreactivity. To analyze whether this assay could also be used for analysis of the early induction phase of the immune response, UCB T cells (n⫽3) or AB T cells (n⫽4) were cocultured with irradiated AML193-DR⫹ cells or AML193-DR⫺ cells, and [3H] thymidine incorporation was measured on day 3. As listed in Table 1, the stimulation indices were low both for UCB T cells (0.8 –1.7) and for AB T cells (0.8 –2.3) when stimulated with AML193-DR⫹ cells or AML193-DR⫺ cells, indicating that [3H] thymidine incorporation was not sensitive enough to measure the limited proliferative response of precursor T cells on day 3 after stimulation. Expression of T Cell Activation Markers CD25 or CD69 After Allogeneic Stimulation Next, we analyzed whether the T cell activation induced by stimulation with the allogeneic APC could be accurately measured by flow cytometric analysis of CD25 or TABLE 1 Stimulation index for UCB T cells or AB T cells on day 3 after stimulation with AML193-DR⫺ cells or AML193-DR⫹ cells Stimulation index Responder
Donor
AML193-DR⫺
AML193-DR⫹
UCB T cells
1 2 3 1 2 3 4
0.8 1 1.4 1.9 1.1 1.7 0.9
0.9 1.2 1.7 0.8 1.2 2.3 1.3
AB T cells
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FIGURE 2 Expression of CD25 or CD69 in unstimulated or stimulated UCB T cells and AB T cells. The percentages of T cells expressing CD25 (A) or CD69 (B) in UCB T cell populations (open symbols; n⫽2 for CD25 and n⫽3 for CD69) and in AB T cell populations (solid symbols; n⫽3 for CD25 and n⫽4 for CD69) after culture without stimulation or after culture with AML193-DR⫹ cells or AML193-DR⫺ cells was measured by flow cytometry on days 1–3 of culture.
CD69 expression on UCB T cells and AB T cells on days 1–3 after stimulation. Figure 2 illustrates the percentages of CD25 or CD69 expressing T cells in UCB T cells or AB T cells when cultured with or without the different stimulator APC. The levels of CD25 expressing T cells in the unstimulated UCB T cells or AB T cells were relatively high on days 1–3 of culture. After stimulation with the different APC no clear increase in the percentages of CD25 expressing T cells in UCB T cells or AB T cells could be detected (Figure 2A). The percentages of CD69 expressing T cells in the unstimulated UCB T cells were low whereas in a number of unstimulated AB T cell fractions relatively high and variable percentages of CD69 expressing T cells were present. The limited changes in percentages of CD69 expressing T cells found for the individual UCB T cell donors or AB T cell donors when stimulated with AML193-DR⫹ cells or AML193-DR⫺ cells were at the detection limit of the flow cytometric method and were highly variable, indicating that the analysis of CD25 or CD69 expressing T cells was not suitable for analyzing the early induction of T cell acti-
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vation on days 1–3 after stimulation with allogeneic APC. Presence of Cytokine-Secreting Cells in UCB T Cell and AB T Cell Populations After Stimulation With AML193-DRⴙ Cells To determine whether allogeneic stimulation under the most optimal circumstances, by optimal APC expressing HLA class I, HLA class II, and costimulatory and adhesion molecules resulted in differential cytokine secretion by naive UCB T cells or AB T cells, the numbers of IL-2–, IFN-␥–, IL-4 –, or IL-10 –secreting T cells on days 1–3 after stimulation with AML193-DR⫹ cells were measured by ELISPOT analysis. The AML193DR⫹ cells did not produce any of the cytokines studied (data not shown). In the unstimulated T cell fractions from UCB, no cytokine-secreting cells were present. The unstimulated AB T cell fractions contained no IL-4 – secreting cells, whereas the numbers of T cells spontaneously secreting IL-2, IFN-␥, or IL-10 ranged from 0 – 4, 0 –27, and 0 –22 per 50,000 cells, respectively. The numbers of cytokine-secreting T cells after stimulation with the AML193-DR⫹ cells were corrected for the numbers of cytokine-secreting cells in the unstimulated T cell populations. AML193-DR⫹ cells did not induce IL-4 secretion in UCB T cells or in AB T cell fractions (0 –3 IL-4 –secreting cells per 50,000 viable T cells). No IL-10 secretion was induced in UCB T cells or AB T cells on days 1 and 2 after stimulation with AML193-DR⫹ cells (data not shown). Three days after stimulation,
Cord Blood T Cells Need HLA-DR for Allo-Activation
FIGURE 3 Numbers of IL-2– or IFN-␥–secreting cells in UCB T cells and AB T cells on days 1–3 after stimulation with AML193-DR⫹ cells. Purified UCB T cells (open symbols; n⫽5) and AB T cells (solid symbols; n⫽5) were stimulated with AML193-DR⫹ cells. On days 1–3 the numbers of IL-2– (A) or IFN-␥– (B) secreting cells per 50,000 viable T cells were determined by ELISPOT analysis. Bars represent medians. Numbers are corrected for the unstimulated T cell populations. *Significant difference between UCB T cells and AB T cells (p ⬍ 0.05).
IL-10 –secreting cells were present at comparable low levels in both UCB T cells and AB T cells ranging from 2–15 and 0 –26 per 50,000 viable UCB T cells and AB T cells, respectively. The numbers of IL-2– or IFN-␥–secreting T cells on days 1–3 after stimulation with AML193-DR⫹ cells are illustrated in Figure 3. AML193-DR⫹ cells induced IL-2 secretion on day 1 in the UCB T cell populations, which did not change on days 2 and 3 after stimulation. In AB T cells, AML193-DR⫹ cells induced IL-2 secretion on day 1, which increased in some cases on days 2 and 3, although the numbers of IL-2–secreting cells between UCB T cells and AB T cells were not statistically different because of the large variation (Figure 3A). AMLDR⫹ cells did not induce IFN-␥ secretion in UCB T cells on day 1 and 2 after stimulation, and on day 3, only limited numbers of IFN-␥–secreting cells were induced. In contrast, AML193-DR⫹ cells induced IFN-␥–secreting cells in AB T cells on days 1–3 after stimulation (Figure 3B).
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FIGURE 4 Numbers of IL-2– or IFN-␥–secreting cells in UCB T cells, and CD45RA⫹ or CD45RO⫹ T cell fractions from AB on days 1–3 after stimulation with AML193-DR⫹ cells. UCB T cells, and CD45RA⫹ or CD45RO⫹ T cell fractions from AB were stimulated with AML193-DR⫹ cells, and the numbers of cells secreting IL-2 (A) or IFN-␥ (B) per 50,000 viable T cells were determined by ELISPOT analysis on days 1–3 after stimulation. Each symbol represents a single donor with open circles for UCB donors (n⫽5), solid circles for CD45RA⫹ T cells from AB (n⫽4), and solid triangles for CD45RO⫹ T cells from AB (n⫽4). Bars represent medians. Numbers are corrected for unstimulated T cell populations. *Significant difference between UCB T cells and CD45RA⫹ T cells from AB; indicates a significant difference between UCB T cells and CD45RO⫹ T cells from AB (p ⬍ 0.05). No statistically significant differences between CD45RA⫹ T cells and CD45RO⫹ T cells from AB were present.
IL-2– or IFN-␥–Secreting Cells in Naive CD45RAⴙ or Memory CD45ROⴙ T Cell Fractions From AB After Stimulation With AML-DRⴙ Cells To analyze whether differences in IFN-␥ or IL-2 secretion between UCB T cells and AB T cells in response to AML193-DR⫹ cells were caused by the presence of memory T cells in AB, or whether there was also an intrinsic difference between the naive CD45RA⫹ T cells from UCB and AB, naive and memory T cell fractions were separated from the AB T cells on the basis of CD45RA or CD45RO expression. In Figure 4, the numbers of IL-2– or IFN-␥–secreting cells present in UCB T cells are compared with the numbers of IL-2– or IFN␥–secreting cells measured in the CD45RA⫹ or the CD45 RO⫹ T cell fractions from AB at days 1–3 after
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FIGURE 5 Numbers of IL-2– or IFN-␥–secreting cells in UCB T cells and AB T cells after stimulation with AML193DR⫹ cells or AML193-DR⫺ cells. On days 1–3 after stimulation with AML193-DR⫹ cells (solid symbols) or AML193DR⫺ cells (open symbols), the numbers of IL-2– (A and C) or IFN-␥– (B and D) secreting cells per 50,000 viable T cells were determined in UCB T cells (n⫽5 for stimulation with AML193-DR⫹ cells and n⫽4 for stimulation with AML193DR⫺ cells) and in AB T cells (n⫽5). Values are corrected for unstimulated controls. *Significant difference between stimulation with AML193-DR⫹ cells and stimulation with AML193-DR⫺ cells.
stimulation with AML193-DR⫹ cells. Values are corrected for the numbers of cytokine-secreting T cells in the unstimulated T cell populations that ranged from 0 –3 and 0 –11 IL-2–secreting cells and 0 –25 and 0 – 66 IFN-␥–secreting cells per 50,000 cells in the CD45RA⫹ and CD45RO⫹ T cell fractions, respectively. In the unstimulated UCB T cells, no IL-2 or IFN-␥ secretion was present. The numbers of IL-2–secreting cells induced by the AML193-DR⫹ cells in naive UCB T cells or naive CD45RA⫹ T cells from AB were similar and levels did not significantly differ on days 1–3 after stimulation. The numbers of IL-2–secreting cells induced by the AML193-DR⫹ cells in the CD45RO⫹ T cell fraction were also low on day 1 but increased significantly on days 2 and 3 after stimulation as compared with UCB T cells (Figure 4A). In contrast, UCB T cells responded to AML193-DR⫹ with only low numbers of IFN-␥–secreting cells on day 3 after stimulation. High numbers of IFN-␥–secreting cells were induced in both the CD45RA⫹ and the
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CD45RO⫹ T cell fractions from AB on days 1 and 2 after stimulation, which was significantly different from UCB T cells (Figure 4B). In conclusion, the difference in IL-2 secretion between UCB T cells and AB T cells in response to stimulation with AML193-DR⫹ cells appeared to be caused by the presence of memory T cells in AB, whereas the difference in IFN-␥ secretion in response to stimulation with AML193-DR⫹ cells was due to a difference in IFN-␥ secretion between naive UCB T cells and both CD45RA⫹ naive T cells and CD45RO⫹ memory T cells from AB. Cytokine-Secreting Cells in T Cell Populations From UCB or AB After Stimulation With AML193-DRⴚ Cells To determine whether the expression of HLA class II on the APC was essential for the induction of cytokine secretion in UCB T cells or AB T cells, the numbers of IL-2–, IFN-␥–, IL-4 –, or IL-10 –secreting cells in UCB T cells or AB T cells on days 1–3 after stimulation with AML193-DR⫹ cells or AML193-DR⫺ cells were compared. Similar to stimulation with AML193-DR⫹ cells, AML193-DR⫺ cells did not induce IL-4 secretion in UCB T cells or AB T cells (data not shown). IL-10 – secreting cells were present on day 3 in both UCB T cells and AB T cells, at comparable low levels ranging from 1–9 and 0 –25 IL-10 –secreting cells per 50,000 UCB T cells and AB T cells, respectively. Figure 5 illustrates the numbers of IL-2– or IFN-␥– secreting cells in UCB T cells or AB T cells after stimulation with the AML193-DR⫺ cells or AML193-DR⫹
Cord Blood T Cells Need HLA-DR for Allo-Activation
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tions were separated from the AB T cells and the frequencies of IL-2– or IFN-␥–secreting T cells were analyzed after stimulation with AML193-DR⫺ cells. The numbers were corrected for the frequencies of cytokinesecreting cells in the unstimulated T cell populations that ranged from 0 –3 and 0 –11 IL-2–secreting cells, and 0 –25 and 0 – 66 IFN-␥–secreting cells per 50,000 CD45RA⫹ T cells and CD45RO⫹ T cells, respectively. As illustrated in Figure 6 , a significant difference in the numbers of IL-2– or IFN-␥–secreting cells in response to AML193-DR⫺ cells between the CD45RA⫹ naive T cells and the CD45RO⫹ memory T cells was only present for the IL-2 secretion on day 1. These results indicate that AML193-DR⫺ cells induced IL-2– or IFN␥–secreting cells in both CD45RA⫹ naive T cells and CD45RO⫹ memory T cells from AB but not in naive UCB T cells.
FIGURE 6 Numbers of IL-2– or IFN-␥–secreting cells in CD45RA⫹ or CD45RO⫹ T cell fractions from AB on days 1–3 after stimulation with AML193-DR⫺ cells. The CD45RA⫹ (open triangles; n⫽4) or CD45RO⫹ (solid triangles; n⫽4) T cell fractions from AB were stimulated with AML193-DR⫺ cells, and the numbers of cells secreting IL-2 (A) or IFN-␥ (B) per 50,000 viable T cells were determined by ELISPOT analysis on days 1–3 after stimulation. Bars represent medians. Numbers are corrected for unstimulated T cell populations. *Significant difference between CD45RA⫹ T cell fractions and CD45RO⫹ T cell fractions.
cells, corrected for the numbers of cytokine-secreting cells present in the unstimulated T cell populations. The background levels in unstimulated AB T cells ranged from 0 – 4 and 0 –27 cytokine-secreting cells per 50,000 cells for IL-2 and IFN-␥, respectively. In UCB T cells, IL-2–secreting cells were induced by AML193-DR⫹ cells but not by AML193-DR⫺ cells. Low numbers of IFN-␥–secreting cells were induced in the UCB T cells by AML193-DR⫹ cells on day 3 of stimulation, but not by the AML193-DR⫺ cells (p ⬍ 0.05) (Figure 5A,B). In contrast, IL-2– or IFN-␥–secreting cells were induced in AB T cells by stimulation with both the AML193-DR⫹ cells and the AML193-DR⫺ cells (Figure 5C,D). In conclusion, UCB T cells but not AB T cells required the expression of HLA class II for the induction of IL-2 secretion and IFN-␥ secretion. To determine whether, similar to naive UCB T cells, IL-2 secretion and IFN-␥ secretion cannot be induced in CD45RA⫹ naive AB T cells by stimulation with AML193-DR⫺ cells, the naive and memory T cell frac-
DISCUSSION The immunological mechanism responsible for the lower incidence and severity of GVHD found for UCB transplantation as compared with adult SCT has not yet been elucidated. In several comparative studies measuring the proliferative, cytotoxic, and cytokine responses of UCB T cells and AB T cells after allogeneic stimulation, a lower alloreactivity was found for UCB T cells [4 –10]. To further investigate the underlying conditions for a reduced alloreactivity in UCB T cells, we analyzed whether UCB T cells and AB T cells have differential requirements for the presence of HLA class II on the APC for the induction of T cell activation, and whether differences were only caused by the presence of memory T cells in AB or also by intrinsic differences between naive T cells from UCB and AB. Because PBMC or cultured DC used as allogeneic stimulator cells are heterogeneous and have variable expressions of HLA class I, HLA class II, and costimulatory and adhesion molecules, we used a cell line with defined and fixed levels of these surface molecules as allogeneic stimulator cells. AML193 cells expressed HLA class I, costimulatory molecules CD86 and CD40 and adhesion molecules ICAM-1 and LFA-3. In addition to the HLA class II–negative AML193 cell line, we used AML193 cells that were induced by IFN-␥ to also express HLADR. The AML193 cell line is derived from a malignant cell and may therefore not reflect all the different APC that are encountered in vivo. Nevertheless, this cell line expressed most of the costimulatory and adhesion molecules that are expressed on maturated APC, and that are important for the generation of an immune response. Therefore, the AML193 cell line provides an adequate model system to study the activation requirements of UCB T cells and AB T cells.
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To analyze the early induction phase of the immune response, we attempted to measure T cell activation on days 1–3 after stimulation of purified T cells from AB or UCB with the different APC. Differences in alloreactivity are often studied by analysis of proliferation by [3H] thymidine incorporation after 5 or more days of in vitro culture [4, 6, 26, 27]. Because 2–3 days after stimulation activated T cells start to rapidly proliferate, analysis later than 3 days of culture will strongly be influenced by the proliferation rate of the responding T cells. However, our results indicate that as early as day 3, [3H] thymidine incorporation was not sensitive enough to accurately measure proliferation of the limited numbers of activated precursor T cells. The analysis of CD25 or CD69 expression by flow cytometry was also found to be not sensitive enough to measure the induction of T cell activation on days 1–3 after stimulation with the different APC. This was in part because of high and variable percentages of CD25 or CD69 expressing T cells present in the unstimulated T cell cultures. Relatively high percentages of CD25 expressing regulatory T cells in unstimulated T cell cultures may be responsible for these findings [28, 29]. The analysis of cytokine-secreting T cells by ELISPOT analysis was found to be sensitive enough to detect T cell activation after 1–3 days of stimulation and was therefore used to further analyze the requirements for the induction of T cell activation in UCB T cells and AB T cells. The analysis of cytokine-secreting T cells was not performed at a later stage than 3 days after stimulation because beyond that time point, rapid cell division is likely to occur. Cell division can strongly influence cytokine secretion, especially during the S-phase of the cell cycle until completion of cell division. Accurate analysis of cytokine secretion at the single cell level can therefore only be performed on days 1–3 after stimulation when extensive cell division is not yet apparent. Because not only the number of activated T cells but also the balance between secreted type 1 cytokines and type 2 cytokines may be important for the clinical outcome of allogeneic responses [30], the numbers of T cells secreting the type 1 cytokines IL-2 or IFN-␥ as well as the numbers of T cells secreting the type 2 cytokines IL-4 or IL-10 were measured. Stimulation with AML193-DR⫹ cells or AML193DR⫺ cells induced a preferential type 1 cytokine response in both UCB T cells and AB T cells with IL-2– or IFN-␥–secreting cells, but almost no IL-4 – or IL-10 – secreting cells. These data are in line with other studies describing the presence of IL-4 – or IL-10 –secreting T cells only after repetitive stimulation with PBMC or DC and not after primary stimulation [4, 8, 31–35] and suggest that IL-4 and IL-10 may only be produced after
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T cell differentiation and not at the initial induction phase of the allo-immune response. Under the most optimal conditions of T cell activation in the presence of both HLA class I and class II on the AML193 cells, IL-2– or IFN-␥–secreting T cells were induced in both UCB T cells and AB T cells. However, the induction of IL-2 secretion and IFN-␥ secretion was slower in UCB T cells, and the numbers of IL-2– or IFN-␥–secreting cells in UCB T cells were lower as compared with AB T cells, indicating a slower kinetics and a lower level of activation in UCB T cells. These data are consistent with studies that describe less secretion of IL-2 or IFN-␥ for UCB T cells as compared with AB T cells after allogeneic stimulation [4, 36]. The differences in kinetics and levels of activation between UCB T cells and AB T cells were not only due to the presence of memory T cells in AB T cells but also due to an intrinsic difference between naive T cells from UCB and from AB. Stimulation of naive CD45RA⫹ T cell fractions and memory CD45RO⫹ T cell fractions with the AML193-DR⫹ cells exhibited an earlier induction and a higher level of IL-2 secretion for the CD45RO⫹ memory T cells but not for the CD45RA⫹ naive T cells from AB, as compared with UCB T cells. In contrast, IFN-␥ secretion was induced earlier and reached a higher level as compared with UCB T cells in both the memory CD45RO⫹ T cells and the naive CD45RA⫹ T cells from AB. For these comparisons with naive UCB T cells, the high purity of the isolated CD45RA⫹ T cell fractions was crucial. The contaminating naive CD45RA⫹ T cells in the memory CD45RO⫹ T cell fractions were found not to significantly influence the outcome as the response induced in memory CD45RO⫹ T cells was higher or comparable to the response induced in naive CD45RA⫹ T cells. On day 1 after stimulation with the AML193-DR⫹ cells, the numbers of IL-2–secreting T cells in the naive CD45RA⫹ T cells and memory CD45RO⫹ T cells were much lower than the numbers of numbers of IFN-␥– secreting T cells in these T cell fractions. This difference is probably due to the presence of large numbers of single T cells that do not produce both cytokines simultaneously but instead produce only one of the two cytokines, with most single T cells secreting only IFN-␥. The presence of such single IL-2– or IFN-␥–secreting T cells in response to different stimuli has also been reported by others [37, 38], and is in agreement with the notion that the separation of T cells into type 1, type 2 and type 0 cytokine-secreting T cells is less strict in humans as compared with mice. The decline in IFN-␥–secreting cells in these two T cell fractions on day 3 after stimulation is most likely caused by the kinetic fashion in which cytokines are produced by single cells [37, 39, 40].
Cord Blood T Cells Need HLA-DR for Allo-Activation
Not only the kinetics and level of activation but also the requirement for HLA-DR expression on the APC for the induction of activation was found to be different for UCB T cells and AB T cells. The expression of HLA-DR on the AML193 cells was found to be essential for the activation of UCB T cells but not for AB T cells, as demonstrated by the virtual absence of IL-2– or IFN-␥– secreting T cells in UCB T cells when stimulated with the AML193-DR⫺ cells whereas the numbers of IL-2– or IFN-␥–secreting T cells present in AB T cells after stimulation with AML193-DR⫺ cells were comparable to the numbers after stimulation with the AML193DR⫹ cells. Because AML193-DR⫺ cells cannot activate CD4⫹ T cells the responses measured after stimulation with the AML193-DR⫺ cells are likely to be derived from the CD8⫹ T cells. Apparently, CD8⫹ T cells from AB but not the CD8⫹ T cells from UCB could respond to stimulation with the AML193-DR⫺ cells. Whether naive CD8⫹ T cells from UCB required the help of CD4⫹ T cells for their activation or whether naive UCB CD8⫹ T cells were completely irresponsive and did also not respond to stimulation with the AML193-DR⫹ cells could not be determined because we did not study CD4⫹ cells and CD8⫹ T cells separately. The capability to respond to APC that did not express HLA-DR was not only found for the memory CD45RO⫹ T cells but also for the naive CD45RA⫹ T cells from AB as both AB T cell fractions secreted IL-2 and IFN-␥ in response to stimulation with the AML-DR⫺ cells. These data further indicated that naive UCB T cells and naive T cells from AB are intrinsically different, with naive UCB T cells but not naive CD45RA⫹ from AB requiring the presence of HLA class II on the APC for allogeneic activation. Furthermore, the observations that the AML193-DR⫹ cells but not the AML193-DR⫺ cells induced cytokine-secreting cells in UCB T cells illustrate that it is highly unlikely that the effects measured in this study are caused by nonspecific stimulation by the AML193 cells. The differences in allo-immune activation between UCB T cells and AB T cells that we found in this study may help to explain the lower GVHD found after UCB transplantation. Naive UCB T cells but not naive or memory T cells from AB may be unresponsive to vascular endothelial cells that under normal circumstances do not express HLA-DR [41] and that are the first APC to be encountered by the donor T cells after transplantation. Even when UCB T cells will be activated by the encounter of APC that express HLA-DR, such as endothelial cells, that have upregulated HLA class II as a result of in vivo inflammatory responses or professional APC like DC, cytokine secretion may be induced more slow in UCB T cells and the level of the cytokine response may be lower as compared with that induced in naive or
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memory T cells from AB, possibly resulting in less severe GVHD. In conclusion, naive UCB T cells but not AB naive T cells require the expression of HLA-DR on the APC for allo-immune activation. Furthermore, in naive UCB T cells allo-immune activation was induced with slower kinetics and at a lower level, as compared with AB naive T cells. These findings may in part explain the reduced incidence and severity of GVHD after UCB transplantation. REFERENCES 1. Rocha V, Cornish J, Sievers EL, Filipovich A, Locatelli F, Peters C, Remberger M, Michel G, Arcese W, Dallorso S, Tiedemann K, Busca A, Chan KW, Kato S, Ortega J, Vowels M, Zander A, Souillet G, Oakill A, Woolfrey A, Pay AL, Green A, Garnier F, Ionescu I, Wernet P, Sirchia G, Rubinstein P, Chevret S, Gluckman E: Comparison of outcomes of unrelated bone marrow and umbilical cord blood transplants in children with acute leukemia. Blood 97:2962, 2001. 2. Rocha V, Wagner Jr JE, Sobocinski KA, Klein JP, Zhang MJ, Horowitz MM, Gluckman E: Graft-versus-host disease in children who have received a cord-blood or bone marrow transplant from an HLA-identical sibling. Eurocord and International Bone Marrow Transplant Registry Working Committee on Alternative Donor and Stem Cell Sources. N Engl J Med 342:1846, 2000. 3. Grewal SS, Barker JN, Davies SM, Wagner JE: Unrelated donor hematopoietic cell transplantation: marrow or umbilical cord blood? Blood 101:4233, 2003. 4. Risdon G, Gaddy J, Stehman FB, Broxmeyer HE: Proliferative and cytotoxic responses of human cord blood T lymphocytes following allogeneic stimulation. Cell Immunol 154:14, 1994. 5. Harris DT, LoCascio J, Besencon FJ: Analysis of the alloreactive capacity of human umbilical cord blood: implications for graft-versus-host disease. Bone Marrow Transplant 14:545, 1994. 6. Roncarolo MG, Bigler M, Ciuti E, Martino S, Tovo PA: Immune responses by cord blood cells. Blood Cells 20: 573, 1994. 7. Keever CA, Abu-Hajir M, Graf W, McFadden P, Prichard P, O’Brien J, Flomenberg N: Characterization of the alloreactivity and anti-leukemia reactivity of cord blood mononuclear cells. Bone Marrow Transplant 15:407, 1995. 8. Slavcev A, Strı¨z I, Ivasˇkova´ E, Breur-Vriesendorp BS: Alloresponses of cord blood cells in primary mixed lymphocyte cultures. Hum Immunol 63:155, 2002. 9. Harris DT, Schumacher MJ, LoCascio J, Besencon FJ, Olson GB, DeLuca D, Shenker L, Bard J, Boyse EA: Phenotypic and functional immaturity of human umbili-
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