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1 Ex vivo Expansion, Maturation and Activation of Umbilical Cord Blood Derived T-lymphocytes with IL-2, IL-12, anti-cd3 and IL-7: Potential for Adoptive Cellular Immunotherapy post Umbilical Cord Blood Transplantation Kathleen L. Robinson, BS 1 *, Janet Ayello, MS 2 *, Rose Hughes, BS 3, Carmella van de Ven, MA 2, Linda Issitt, MT (ASCP) SBB 4, Joanne Kurtzberg, MD 4, and Mitchell S. Cairo, MD 2 1 Department of Pediatrics, Georgetown University, Washington, DC 2 Children's Hospital of New York, Columbia University, New York, New York 3 Beckman Coulter, Inc., Miami, Florida 4 Department of Pediatric Bone Marrow Transplantation, Duke University, Durham, North Carolina Presented in part at the American Society of Hematology (ASH), December 1999, New Orleans, Louisiana. Supported in part by grants from the Pediatric Cancer Research Foundation and the Phi Beta Psi Sorority Address Inquiries to: Mitchell S. Cairo, M.D. Director, Pediatric Blood and Marrow Transplantation Children s Hospital of New York Columbia University 161 Fort Washington, Irving 7 New York, New York Phone: Fax: mc1310@columbia.edu Table of Contents Category: Lymphopoiesis or Cytokines Word Count: 3730 * Both contributed significantly to this manuscript and should be considered co-first (primary) authors

2 ABSTRACT Objectives: We investigated whether umbilical cord blood (UCB) T cells could be ex vivo expanded and activated in short term culture for potential utilization as adoptive cellular immunotherapy post umbilical cord blood transplantation (UCBT). Methods: Fresh UCB mononuclear cells (MNCs) were isolated by ficoll density centrifugation. Cryopreserved UCB mononuclear cells were thawed and washed with 2.5% human serum albumin and 5% dextrose in isotonic saline. The nonadherent MNC fraction were then plated in a serum-free cocktail of IL-2, IL- 12, and anti-cd3 with and without IL-7 for 48 hours. Proliferation, cytotoxicity, TH1 (IFNγ), CD25, and CD45RO assays were performed. Results: Proliferation studies demonstrated a significant increase in the proliferative ability of the UCB MNCs incubated in anti-cd3, IL-2, IL-12, and IL-7 (fresh--p<0.005 and thawed--p<0.001). The combination of all four agonists significantly induced expression of CD45 RO (fresh--p<0.05, and thawed--p<0.001) in both the CD4+ and CD8+ T cells expressing CD25 (fresh UCB--p<0.01 [CD4] and p<0.005 [CD8], respectively; thawed UCB-- p<0.001 [CD4] and p<0.001 [CD8]). Intracellular cytokine profiles also revealed a significant increase in the production of IFN-γ (TH1 cells) (fresh UCB--p<0.005, and thawed UCB--p<0.001). The combination also significantly increased the killing of K562 labeled target cells (fresh--p<0.0001, and thawed ± 0.03 vs ± 0.01) (p<0.001). Conclusion: These data suggest that the ex vivo combination of IL-2, IL-12, anti-cd3, and IL-7 significantly enhances the proliferation, activation, maturation and cytotoxic potential of UCB T cells of both fresh and thawed UCB MNC. Further studies, however, are required to determine whether these ex vivo expanded MNC could also potentially exacerbate acute or chronic graft-vs-host disease and/or other toxicities if utilized post UCBT. Keywords: Umbilical cord blood, donor lymphocytes, ex vivo expansion, IL-2, IL-12, IL-7, anti-cd3, cytotoxic T-lymphocytes.

3 INTRODUCTION The recent increase in the number of umbilical cord blood (UCB) units cryopreserved and the suggestion of a decreased incidence of severe (grade III/ IV) acute graft-vs.-host disease (GVHD) following umbilical cord blood transplantation (UCBT) compared to similar unrelated bone marrow HLA disparate allografts have contributed to the increased use of unrelated UCB as an alternative source of allogeneic stem cells for the treatment of both malignant and non malignant disorders [1-6]. Studies in our laboratory have reported a delay in T-cell reconstitution following unrelated UCBT which in part could account for the high rate of infectious morbidity seen post UCBT [7]. Several limitations are associated with using unrelated UCB as an alternative source of allogeneic stem cells for hematological malignancies, including the delay in T-cell reconstitution and, more importantly, the lack of available donor immuno-effector cells for adoptive cellular immunotherapy post UCBT to treat minimal residual disease, recurrent disease, or post-transplant lymphoproliferative disease [3]. It has previously been shown that UCB T cells are phenotypically and functionally immature with a higher percentage of CD45RA+ T cells and decreased numbers of alloantigen-specific cytotoxic T lymphocytes (CTLs) [8-11]. Further, several investigators have reported that neonatal T cells have a reduced capacity to produce cytokines. Wilson et al. [12] reported a 10-fold decrease in the production in interferon-γ (IFN-γ) in neonatal compared with adult T cells, and English et al. [13] demonstrated a 50% decrease in the levels of TNF in neonates compared to adults. We have previously demonstrated that UCB mononuclear cells also produce lower levels of IL-12 and IL-15 evidenced by a significantly decreased mrna half-life and decreased protein production following activation when compared to adult peripheral blood (APB) [14, 15]. Chalmers et al. have further confirmed that UCB lymphocytes stimulated with PMA and inomycin in the presence of monensin produced lower intracellular levels of IL-2, IL-4, IFN-γ and TNF-α compared to similarly-treated adult peripheral blood lymphocytes [16]. More recently, reports of aberrant signaling in UCB T cells due to decreased phospholipase C activation, low levels of Lck [17], and decreased levels of the transcription factor NFAT1 [18] have been reported. In summary, UCB T- cell immunity is immature with respect to alloantigen recognition, lymphokine production, and T cell activation compared to mature adult peripheral blood T cells. In order to circumvent these above mentioned limitations of UCBT, we investigated a novel strategy for the ex vivo expansion and activation of UCB T cells for potential adoptive cellular immunotherapy. The ex vivo expansion of T lymphocytes with the combination of anti-cd3 and IL-2 has been used in adoptive cancer-based cellular immunotherapy and vaccine trials [19, 20]. Alvarnas et al. reported the ex vivo expansion of CD3+/CD56+ lymphocytes from adult peripheral donors after priming with IFN-γ, anti-cd3 monoclonal antibody followed by IL-2 [21]. IL-12 has been used to generate autologous CTLs [22] and to induce proliferation of UCB-derived MNCs [23]. IL-7 has been shown to be necessary for T-cell development and can promote T-cell proliferation, especially in CD8+ T cells [24, 25]. We examined the use of cytokines (IL-2, IL-12 and IL-7) and specific monoclonal antibodies (anti-cd3) in the ex vivo generation of mature and activated T cells from fresh and previously cryopreserved UCB mononuclear cells. MATERIALS AND METHODS Umbilical Cord Blood Collection: After obtaining maternal donor consent, fresh blood was collected from the umbilical cord vein using the method previously described in the National Heart, Lung and Blood Institute (NHLBI) for Cord Blood Transplantation (COBLT) study [26]. The various collection centers local institutional review boards approved this research protocol. Briefly, after aseptic preparation of the cord, the umbilical vein was punctured using a sterile 18-gauge needle attached to sterile tubing on a 200 ml sterile collection bag. The bag contained 25 ml of CPD (citratephosphate-dextrose) as an anticoagulant. Cord blood was collected into a bag by gravity flow with constant rocking. Similarly, additional UCB was RBC depleted, cryopreserved and thawed according to the methods previously described by the NHLBI/COBLT protocol [26]. Mononuclear Cells Isolation and Culture: 2

4 Fresh UCB was layered over Ficoll-Paque TM Plus (Amersham, Arlington Heights, IL) and centrifuged at 400 x g with the brake off for 30 minutes. MNCs at the interface were collected and washed in phosphate buffered saline (PBS) (Gibco, Grand Island, NY). Cryopreserved UCB was thawed and washed according to the method of Rubinstein et al [4]. Briefly, the UCB units were thawed at 37 C for 2 min. The UCB was diluted 1:1 with Dextran/human serum albumin wash (2.5% HAS and 5% dextrose in isotonic saline). It was mixed and centrifuged at 400 x g for 10 min. The supernatant was removed and the cells resuspended slowly in the dextran/albumin wash in equal volume. Both fresh and thawed UCB cells were resuspended at 5x10 6 cells/ml in human cord blood media-2 (HCBM-2) (Aim V media [Gibco] + 20 U/ml heparin [Elkins-Sinn, Cherry Hill, NJ] + 50 µm/ml 2-mercaptoethanol [Gibco]) and incubated overnight in a 5% CO 2 37 C humidified incubator to deplete the adherent monocytes. The non-adherent cells were removed. The fresh CB underwent a second ficoll procedure to remove any nucleated red blood cells. The lymphocyte interface was washed in PBS (Gibco). Viability of the fresh and thawed cells was confirmed by staining with trypan blue (Gibco). The cells of both conditions were separately placed in culture at 1x10 6 cells/ml in T25 flasks (Costar, Cambridge, MA). The cytokines were added as follows: 1) HCBM-2 alone; 2) HCBM-2 supplemented with 5 ng/ml IL-2 (Peprotech, Rocky Hill, NJ), + 10 ng/ml IL-12 (Peprotech), + 50 ng/ml anti-cd3 (Pharmingen, San Diego, CA); or 3) HCBM-2 supplemented with 5 ng/ml IL-2, + 10 ng/ml IL-12, + 50 ng/ml of anti-cd3, + escalating doses of IL-7 at 1, 10, or 100 ng/ml (Peprotech). Cells were cultured for 48 hours at 37 C, 5% CO 2. Viability and Proliferation Testing: After 48 hours, viability was determined by trypan blue (Gibco) staining. Proliferation was assessed by tetrazolium degradation of WST-1 determined with the standard kit available from Boehringer Mannheim (Indianapolis, IN). In brief, 10 µl of WST-1 dye was added to 100 µl of the UCB MNC cell cultures in a 96-well flat bottom plate (Costar) and incubated for three hours. The plate was shaken for 1 minute and the absorbance was read at 450 nm by the plate reader (Beckman, Fullerton, CA). Flow Cytometry: 5x10 5 cells were resuspended in 50 µl of azide buffer (10% FA Bacto Buffer [Difco, Detroit, MI], 1% sodium azide [Sigma, St. Louis, MO], 10% heat inactivated FBS [Gibco]) for evaluation. Fluorescent conjugated monoclonal antibodies CD3, CD4, CD8, CD25 and CD45RO (Coulter, Hialeah, FL) were added to the cells and incubated on ice for 15 minutes. The cells were then washed in azide buffer and fixed with 2% paraformaldehyde (Sigma). Isotype controls were run with each experiment. Fresh UCB samples were read on a Coulter Epics XL-MCL (Coulter). Thawed UCB samples were read on a Becton Dickinson FACS Calibur (Becton Dickinson Immunocytometry, San Diego, CA). The CD3+ population was gated and used as a reference for the determination of CD4, CD8, CD25 and CD45RO populations. A minimum of 5000 events was collected for the fresh UCB samples for the CD45RO staining while 10,000 events were collected for other parameters and experiments. Cytokine Staining: To block IFN-γ release from the cell membrane into the supernatant before staining, cell cultures were incubated for four hours with 1 µg/ml Brefeldin A (Boehringer Mannheim, Indianapolis, IN). The cells were then stained for extracellular CD3, fixed and permeabilized by the IntraprepTM Permeabilization Reagent (Coulter). The cells were next incubated with IFN-γ FITC monoclonal antibody (Coulter). The cells were next washed in azide buffer and fixed in 2% paraformaldehyde/azide buffer. Control samples were stained only for CD3, not permeabilized or stained with the IFN-γ FITC monoclonal antibody. All samples, fresh and cryopreserved, were read within 24 hours on the Coulter Epics XL-MCL and Becton Dickinson FACS Calibur, respectively. The CD3 positive population was gated and served as a reference to determine the intracellular IFN-γ staining. A minimum of 10,000 events was collected for each experiment. Cytotoxicity Assays: Fresh Cord Blood: Cellular cytotoxicity was determined by a modified europium assay measured by the release of BATD from labeled target cells, which forms a fluorescent chelate with europium [27]. K562 target cells were resuspended in cytotoxic T-lymphocyte (CTL) media (500 ml Iscove s media [Gibco] + 5% fetal calf serum + 5 ml L-glutamine [Gibco] + 5 ml Pen/Strep [Gibco], + 5 ml of sulfinpyrazone [SPZ] solution [Sigma] [0.7 3

5 g SPZ ml PBS] ml PBS [Gibco] ml 10N NaOH]) and incubated with 5 µl/ml of BATD-A (Wallac, Gaithersburg, MD) for 30 minutes. The targets were washed three times with CTL media and resuspended at a final concentration of 1x10 5 cells/ml. Target cells were transferred to the appropriate wells of a 96-well round bottom plate (Costar). Effector cells were added to target cells in experimental wells at a 20:1 effector-to-target ratio. Control wells contained 100 µl of the labeled target cells and either 100 µl of CTL media to measure spontaneous release or 100 µl of 2% Triton (Sigma) to measure maximum release. The plate was placed in a 5% CO 2 37 C humidified incubator for a 90-minute incubation. Plates were then centrifuged and 50 µl of the supernatant was transferred to a 96-well flat-bottomed plate (Costar) containing 200 µl of europium buffer. The plates were shaken for 5 minutes and the absorbance was read by time resolved fluorometry on the VictorII Plate reader (Wallac). Percent cytotoxicity was determined by the standard equation (experimental release-spontaneous release/ maximum release-spontaneous release x 100). All experiments were performed in triplicate. Cryopreserved Cord Blood: Cellular cytotoxicity assay was determined by the metabolization of MTT determined with the standard kit (Roche Molecular Biochemicals, Mannheim, Germany). K562 cells were washed with CTL media and resuspended at a final concentration of 1 x 10 5 cells/ml. Effector cells were added to target cells in experimental wells at 20:1 effector-to-target ratio. Control wells contained 100 µl of the effector:target cell culture in a 96-well flat bottom plate (Costar) and incubated for 4 hours in 5% CO 2 at 37 C. 100 µl of solubilization solution was added to each well and incubated overnight in 5% CO 2 at 37 C. The plate was shaken for one minute and the absorbance read at 500 nm by a plate reader (Beckman, Fullerton, CA). All experiments were performed in seven. Statistical Analysis: Results are presented as mean ± standard error of the mean. The probability of significant differences when comparing two groups was determined with the use of a two-tailed unpaired Student s T test. The probability of significant differences when comparing multiple treatment groups was determined by the analysis of variance followed by the Tukey s multiple range test. Statistical analyses were performed using the Instat statistical program (Graph Pad, San Diego, CA). P values <0.05 are considered significant. RESULTS Proliferation Assay: The proliferation of the non-adherent UCB MNCs after 48 hours in culture was determined by the WST-1 proliferation assay. Fresh UCB cells cultured in IL-2 + IL-12 + anti-cd3 showed no significant increase when compared to cells incubated in HCBM-2 media alone (data not shown). However, thawed cryopreserved UCB cells showed a significant increase with this cocktail (0.18 ± 0.02 vs ± 0.02%) (p<0.001) (media vs. anti-cd3/il-2 and IL-12) (n=7) (Table 1). The cultures containing escalating doses of IL-7 showed increased proliferation (Figure 1). The addition of IL-7 to IL-2 + IL-12 + anti-cd3 showed significant increased fresh UCB proliferation when compared to IL-2 + IL-12 + anti-cd3 alone at 10 and 100 ng/ml (1.10 ± vs ± 10 ng/ml, p<0.05; 1.15 ± vs ± 100 ng/ml, p<0.005; n=3). Since optimal proliferation appeared to be reached at 10 ng/ml IL-7, it was decided to use that concentration in subsequent experiments. Utilizing thawed previously cryopreserved UCB, the addition of IL-7 to anti-cd3, IL-2 and IL-12 also significantly increased proliferation compared to media alone and the cocktail without IL-7 (0.67 ± 0.03 vs ± 0.02% or vs ± 0.02%) (p<0.001 and p<0.001, respectively, n=7) (Table 2). CD45RO Expression Assay: Next we investigated the effect of the addition of IL-7 to the cytokine combination on CD45RO isoform expression in the CD3+ T cell population. We noted a significant increase in the CD3+ population in the thawed UCB cultures containing IL-2 + IL-12 + anti-cd3 + IL-7 compared to media alone (3.25 ± 0.49 x 10 6 vs ± 0.07 x 10 6, p<0.01, n=7). Further, the addition of IL-7 to cultures containing IL-2 + IL-12 + anti-cd3 significantly increased the numbers of CD3+ cells compared to expansion without IL-7 (7.37 ± 0.82 x 10 6 vs ± 0.49 x 10 6, p<0.001, n=7). CD45RO expression on CD3+ T cells incubated in IL-2 + IL-12 + anti-cd3 + IL-7 showed a 4

6 significant increase in CD45RO isoform expression compared to those cells incubated in HCBM-2 media alone (fresh UCB: 33.1 ± 4.34% vs ± 0.456%, p< 0.005, n=4; thawed UCB: 38 ± 2.4 vs. 2.6 ± 0.57%, p<0.001, n=7). There was, however, no difference between fresh and thawed UCB CD45RO expression (Table 1). CD25 Expression Assay: We also evaluated the expression of CD25, the IL-2 receptor, in proliferating CD4+ and CD8+ T-cell populations by extracellular flow cytometry. To confirm that the test sub-populations of T cells were not non-specifically stimulated, we examined and determined that the CD4+ T cells incubated in HCBM-2 media alone showed the equivalent expression of CD25 as the CD4+ T cells before our culture process (data not shown). However, after 48 hours, cells cultured with IL-2 + IL-12 + anti-cd3 with and without additional IL-7 showed a significant increase in the CD4+/CD25+ expression when compared to cells cultured in HCBM-2 media alone (fresh UCB: ± 2.66% vs ± 0.51%, p<0.0001; ± 4.83% vs ± 0.51%, p<0.0001; n=6 [Table 2]; thawed UCB: 55.5 ± 2.3 vs ± 0.4%, p<0.001; 29.4 ± 1.3 vs ± 0.4%, p<0.001; n=7). Also, cultures containing IL-2 + IL-12 + anti-cd3 with and without IL-7 showed a significant increase in CD8+/CD25+ expression when compared to cells incubated in HCBM-2 media alone (fresh UCB: ± 4.19% vs ± 0.009%, p<0.003; 8.96 ± 3.44% vs ± 0.009%, p<0.03; n=6 [Table 2]; thawed UCB: 16.2 ± 0.58 vs ± 0.13%, p<0.001, 7.21 ± 0.78 vs ± 0.13%, p<0.001; n=7). TH1 Cells (IFNγ Expression) Assay: Comparable levels of IFN-γ were observed before culture (1.63 ± 0.417) and after incubation in HCBM-2 media alone (2.88 ± 0.797, p=ns, n= 6). CD3+ T cells cultured in IL-2 + IL-12 + anti-cd3 with and without IL-7 showed significant increases in levels of IFN-γ compared to cells before culture (fresh UCB: ± 2.00% vs ± 0.797%, p<0.005; 7.41 ± 1.06% vs ± 0.797%, p<0.005; n=6 [Figure 2a]; thawed UCB: 9.39 ± 0.48 vs ± 0.54%, p<0.001; 7.99 ± 0.88 vs ± 0.54%, p<0.05; n=7, [Figure 2b]). However, there was no significant difference in the upregulation of IFN-γ in cells cultured with IL-2+ IL-12 + anti-cd3 vs. IL-2+ IL-12 + anti-cd3+ IL-7 (both fresh and thawed). Cytotoxicity Assay: Cells incubated in IL-2 + IL-12 + anti-cd3 and IL-7 exhibited a significant increase in cytotoxicity compared to cells cultured in HCBM-2 media alone or without IL-7 (fresh UCB: ± 1.72 vs ± 0.024%, p<0.0001, and ± 1.72 vs ± 9.62%, p<0.05, n=4 [Figure 3a]; thawed UCB: ± vs ± 0.01, p<0.001, and ± 0.035% vs ± 0.026%, p<0.001, n=7 [Figure 3b]). DISCUSSION One of the limitations following unrelated UCBT has been the inability to treat hematological malignant relapse and/or post-transplant lymphoproliferative disease (PTLD) with donor leukocyte infusions (DLI) post transplantation because of the lack of available DLI after UCBT. Locatelli et al. demonstrated a 10-20% incidence of leukemia relapse after UCBT in children treated for acute leukemia [28]. Barker et al. demonstrated 5 cases of PTLD in 272 UCBT recipients.[3] DLI has been used successfully following UCBT for CML because of the availability of DLI from the sibling donor peripheral blood [29]. Furthermore, IL-2 has been successfully utilized post UCBT in an AML recipient who developed acute GVHD and a graft-vs-leukemia (GVL) effect and induced disappearance of subcutaneous nodules [30]. These results suggest that if safe and effective methods were available to ex vivo generate DLI from the original aliquot of cord blood, then treatment of hematological relapse and/or PTLD could be made possible in the future following unrelated UCBT. The combination of IL-2 + IL-12 + anti-cd3 ± IL-7 demonstrated an increased ability to mature and activate UCB-derived T cells ex vivo compared to cells incubated in media alone. However, the addition of IL-7 to IL-2 + IL-12 + anti-cd3 further significantly enhanced the levels of proliferation and cytotoxic killing compared to cells incubated in media alone and with IL-2, IL-12 and anti-cd3 from both fresh and thawed UCB specimens. Previous reports indicate that stimulating T cells with PHA + anti-cd28 + IL-7 increases the DNA binding activity of the transcription factor NFAT by 60% [31]. Our addition of IL-7 into this cocktail may in part have helped 5

7 compensate for the decreased levels of NFAT reported in UCB T cells because the increased binding activity could compensate for the decreased levels observed [18]. After 48 hours in culture, the CD3+ cells showed a significant CD45RO isoform switch indicating the generation of memory cells by this ex vivo cocktail with both fresh and cryopreserved specimens. The loss of CD45RA+ isoform and the subsequent gain of the CD45RO+ isoform has previously been demonstrated [10, 32]. UCB T cells following allostimulation showed a marked decrease in their CD45RA levels by flow analysis [10]. Newborn isolated CD4/CD45RA+ T cells stimulated with PHA or anti-cd2 were also capable of switching to the CD45RO isoform [32]. We further observed a significant increase in the expression levels of CD25, a lymphocyte activation marker, in both the CD4 and CD8 subpopulations, after the 48-hour incubation of UCB MNC with the cocktail containing IL- 2 + IL-12 + anti-cd3 + IL-7 in both fresh and thawed specimens. Armitage et al. previously reported proliferation in adult peripheral blood derived T cells with IL-7 + anti-cd3, IL-7 + lectin, or IL-7 alone [24]. All three conditions gave rise to the upregulation of CD25 in the CD4+ and CD8+ populations [24] and is consistent with what we observed in UCB T cells. Of additional interest to us was the effect of the combination of IL-2 + IL-12 + anti-cd3 + IL-7 on the Th1 population of T cells. Th1 T cells are characterized by their cytokine profile, most notably the release of IFN-γ. The number of T cells expressing IFN-γ significantly increased during our ex vivo incubation, leading us to believe that this cocktail is capable of inducing the formation of Th1 T cells in both fresh and thawed specimens. This is supported by previous reports that UCB T cells, both those CD45RA+ and CD45RO+, can produce IFN-γ when stimulated with PMA and ionomycin [16]. Enhancement of cytotoxicity was demonstrated by incubation with the IL-2 + IL-12 + anti-cd3 + IL-7 cocktail determined by both the europium release cytotoxicity assay and MTT assay in fresh and thawed UCB, respectively. Sun et al. reported that antigen-specific UCB CTLs could be generated against Epstein-Barr virus [33]. Their studies demonstrated that UCB MNCs stimulated with transformed cell lines and IL-2 for a period of 28 days generated CD4+ cells that were able to demonstrate specific killing by a perforin/granzyme B dependent manner. Joshi et al. have also demonstrated that UCB mononuclear cells stimulated with IL-2 for 72 hours have an increased cytotoxicity of both K562 and Raji cells in vitro compared to similarly-treated adult peripheral blood cells [34]. These stimulated UCB mononuclear cells also have an increased capacity in vitro to kill the breast cancer cell line MDA-231 [34]. Our findings are consistent with these results and suggest that UCB CTLs can be generated in vitro for a wide range of cellular immunotherapy. In summary, we have shown that the ex vivo combination of IL-2, IL-12, anti-cd3 and IL-7 can successfully activate and induce maturation in UCB-derived T lymphocytes in both fresh and previously-cryopreserved UCB. This cytokine/antibody ex vivo approach to expand and activate cytotoxic T lymphocytes may play a potential role in ex vivo adoptive cellular immunotherapy after cord blood transplantation. However, further studies are needed to determine the safety and efficacy of this ex vivo adoptive cellular immunotherapy approach, especially the risks of acute or chronic GVHD and its associated morbidity. Acknowledgements: The authors would like to acknowledge Elizabeth Joyal, RN, and Ellen Areman, MA, for their assistance in obtaining cord blood specimens during the NHLBI/ COBLT study. The authors would also like to acknowledge Linda Rahl for her assistance in the preparation of this manuscript. 6

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10 Table 1 Comparison of UBC MNC proliferation and CD45RO expression between fresh and thawed UCB ex vivo expanded with anti-cd3/il-2/il-12/il-7 or media alone Proliferation Fresh Cord Blood Thawed Cord Blood Media 0.18 ± ± 0.02 Anti-CD3/IL-2/IL-12/IL ± ± 0.03 P value < < CD45RO Expression (%) Media 1.69 ± ± 0.57 Anti-CD3/IL-2/IL-12/IL ± ± 2.35 P value < < P value -- media vs. anti-cd3/il-2/il-12/il-7 9

11 Table 2 Comparison of UCB CD4/CD25 and CD8/25 expression between fresh and thawed UCB ex vivo expanded with anti-cd3/il-2/il-12 ± IL-7 or media alone CD4/25 (% expression) Fresh Cord Blood Thawed Cord Blood Media 3.88 ± ± 0.43 Anti-CD3/IL-2/IL ± 4.83 * ± 1.25 * Anti-CD3/IL-2/IL-12/IL ± 2.66 ** ± 2.29 ** P value (vs. media) *p<0.001, **p<0.001 *p<0.001, **p<0.001 CD8/25 (% expression) Media ± ± 0.13 Anti-CD3/IL-2/IL ± 3.44 * 7.21 ± 0.78 * Anti-CD3/IL-2/IL-12/IL ± 4.19 ** ± 0.58 ** P value (vs. media) *p<0.03, **p<0.003 *p<0.001, **p<

12 FIGURES Figure nm IL-2 + IL IL-7(1ng/ml) + IL-7(10ng/ml) + IL-7(100ng/ml) anti-cd3 Proliferation of the non-adherent fresh umbilical cord mononuclear cells after 48 hours in culture as determined by the WST-1 proliferation assay. Cells were incubated with either IL-2, IL-12 + anti-cd3; or IL-2, IL-12, anti-cd3 + escalating doses of IL-7 (1, 10, 100 ng/ml). Results represent the mean ± sem of three separate experiments; p<0.005, IL-7 (10 ng/ml) vs. control; p<0.05, IL-7 (100 ng/ml) vs. control. Figure 2 a 11

13 14 12 p<0.005 % of IFN-g/ CD3 (+) Cells p< Media Alone IL-2 + IL-12 + anti-cd3 Day 2 IL-2 + IL-12 + anti-cd3 + IL-7 b 10 p<0.001 p<0.05 % of IFN-g/CD3 cells (%) Media Alone IL-2 + IL-12 + anti-cd3 Day 2 IL-2 + IL-12 + anti-cd3 + IL-7 Figure 2 a and b: Level of IFN-γ present in stimulated fresh or thawed UCB T cells as determined by intracellular flow cytometry. IFN-γ release was blocked before staining with Brefeldin A then stained for CD3 expression. The cells were then fixed, permeabilized and incubated with IFN-γ-FITC monoclonal antibody. CD3+ population was gated and used as reference for intracellular IFN-γ staining. The bars represent: cells that were 12

14 unstimulated day 0; cells day 2, media alone; cells day 2, IL-2, IL-12 + anti-cd3; cells day 2, IL-2, IL-12, anti- CD3 + IL-7. A minimum of 10,000 events was collected for evaluation. Results represent the mean ± sem of six or seven experiments, respectively. Fresh cord blood (Figure 2a) p<0.005, IL-2 + IL-12 + anti-cd3 vs. media; p<0.005, IL-2 + IL-12 + anti-cd3 + IL-7 vs. media; thawed cord blood (Figure 2b) p< 0.05, IL-2 + IL-12 + anti- CD3 vs. media, p< 0.001, IL-2 + IL-12 + anti-cd3 + IL-7 vs. media. Figure 3 a 60 p< p< % Cytotoxicity p< Media Alone IL-2 + IL-12 + anti-cd3 IL-2 + IL-12 + anti-cd3 + IL-7 b 0.8 p< nm-690 nm p< p< Media Alone IL-2 + IL-12 + anti-cd3 IL-2 + IL-12 + anti-cd3 + IL-7 13

15 Figure 3 a and b: Cellular cytotoxicity of stimulated fresh and thawed UCB T cells determined by a modified europium assay measured by the release of BATD from labeled K562 target cells or metabolization of MTT, respectively. Effector cells were added to the target cells in the experimental wells at a 20 to 1 effector-totarget ratio. Bars represent % cytotoxicity or absorbance (A 550nm -A 690nm ) of cells stimulated with: media alone, IL- 2, IL-12 + anti-cd3; IL-2, IL-12, anti-cd3 + IL-7. Results represent the mean +/- sem of four or seven separate experiments, respectively. Fresh cord blood (Figure 3a) p<0.05, IL-2 + IL-12 + anti-cd3 vs. media; p<0.05, IL-2 + IL-12 + anti-cd3 + IL-7 vs. IL-2 + IL-12 + anti-cd3; p<0.0001, IL-2 + IL-12 + anti-cd3 + IL-7 vs. media. Thawed cord blood (Figure 3b) p<0.01, IL-2 + IL-12 + anti-cd3 vs. media; p<0.001, IL-2 + IL-12 + anti-cd3 + IL-7 vs. IL-2 + IL-12 + anti-cd3; p<0.001, IL-2 + IL-12 + anti-cd3 + IL-7 vs. media. 14