WO2000044893A1

WO2000044893A1 - In vitro activated gamma delta lymphocytes

Info

Publication number: WO2000044893A1
Application number: PCT/US2000/001867
Authority: WO
Inventors: Lawrence S. Lamb, Jr.
Original assignee: PALMETTO HEALTH ALLIANCE D/B/A PALMETTO RICHLAND MEMORIAL HOSPITAL
Current assignee: PALMETTO HEALTH ALLIANCE D/B/A PALMETTO RICHLAND MEMORIAL HOSPITAL
Priority date: 1999-01-28
Filing date: 2000-01-27
Publication date: 2000-08-03
Anticipated expiration: 2001-07-28
Also published as: AU771710B2; AU3472800A; JP2002535002A; IL144339A0; EP1147186A1; CA2360046A1; WO2000044893A9; EP1147186A4

Abstract

Patients who develop increased numbers of ηδ+ cytotoxic T lymphocytes 2-6 months after allogeneic bone marrow transplantation are less likely to relapse than those who do not. The ηδ+ T cells isolated from blood of patients with increased ηδ+ T cells are CD3+CD4-CD8-CD57+, cytolytic to K562 cells, and express the Vδ1 T cell receptor phenotype. Similar ηδ+ T cells can be generated in vitro by culture of donor mononuclear cells which are enriched for ηδ+ T cells by immunomagnetic depletion of depleted of CD4+ and CD8+ cells. This ηδ-enriched cell preparation was cultured on a combination of immobilized pan-δ monoclonal antibody and irradiated recipient B cell leukemia. After four weeks, the cultures were almost exclusively Vδ1+CD3+CD4-CD8- cells that co-expressed activation-associated antigens CD69, CD25, and HLA-DR. Furthermore, they were cytolytic against the primary leukemia obtained from the recipient and lymphoblastic leukemia cell lines, yet had minimal cytotoxicity against normal donor-derived mononuclear cells or myeloid leukemia cell lines. These observations suggest that donor-derived cytotoxic ηδ+ T cells can be generated in vitro, and may provide therapeutic potential for prevention of disease relapse.

Description

IN VITRO ACTIVATED GAMMA DELTA LYMPHOCYTES BACKGROUND OF THE INVENTION

Allogeneic bone marrow transplantation (BMT) provides a potentially curative treatment for leukemias that are refractory to conventional therapy. In addition to providing hematopoietic rescue from myeloablative therapy, BMT offers an adoptive immunotherapy effect (graft-versus-leukemia-GvL) that can be beneficial in the elimination of residual leukemia. This was initially shown in cases where T cell depletion (TCD) has been used to prevent graft-versus-host disease (GvHD) but also experienced an increase in disease relapse (1). Indeed, relapse rates in high-risk patients (long-standing recurrent disease or relapse at the time of BMT) can be as high as 70% (2). Therefore, further improvement in disease- free survival is likely to depend on the antileukemic effectiveness of the transplant, i.e. maximizing the GvL effect.

Most experimental evidence suggests that GvL effectors are predominantly T cells that can either recognize allospecific molecules expressed on both normal and neoplastic hematopoietic cells or recognize cell surface molecules that are either unique to or preferentially expressed by the leukemia (3-7). Identification of specific cell populations that are important antileukemic effectors is an essential first step to successful GvL graft engineering and cellular immunotherapy. Although several studies have suggested that γδ+ T cells may not be important primary effectors of GvHD (8-12), few have addressed the GvL potential of γδ+ T cells. Esslin (13) noted that in vitro activated γδ+ T cells can mediate broadly-based non-MHC restricted cytolytic activity to selected human tumor cell lines. Others have shown that γδ+ T cells can recognize unprocessed peptides, some of which are preferentially expressed on tumor cells (14-18). Finally, one report has shown cytotoxic anti-leukemic activity in a patient against B cell ALL by γδ+ T cells expressing the Vδ1 form of the T cell receptor (19). Taken together, these findings support a potential antileukemic role for γδ+ T cells.

Published data describing a series of 10 leukemia patients who developed an increased proportion of circulating CD3+CD4-CD8-Vδ1+γδ+ T cells between 60 and 270 days post-BMT from a partially mismatched related donor (PMRD) which continued for up to two years. Eight of these patients are surviving and remain free of disease, as compared to a DFS probability of 31 % at 2.5 years among 100-day survivors with a normal number of γδ+ T cells (20). In addition, it has been recently shown that enrichment of the graft with γδ+ T cells may have contributed to the later development of increased γδ+ T cells (21). Regardless of the TCD protocol used, however, patients who developed increased γδ+ T cells showed the same cell phenotype and cytolytic function as well as a decreased incidence of relapse. Allogeneic Bone Marrow Transplantation and Graft-Host Interactions:

High-dose chemo/radiotherapy followed by bone marrow rescue provides a potentially curative treatment for a variety of leukemias and solid tumors that are refractory to conventional therapy. An alloreactive response, mediated by donor immunocompetent cells in the graft and directed against normal cells and tissues in the recipient can result in the development of graft-versus-host disease (GvHD). GvHD can occur in up to 50% of patients receiving unmodified, HLA-identical marrow, indicating that minor histocompatibility differences, not detected by conventional HLA matching techniques, can initiate this reaction (22,23). For the majority of patients (approximately 70%) who do not have matched sibling donor (MSD) alternative donors may be used but the risk of acute GvHD is increased due to differences in major as will minor histocompatibility antigens (1). The same alloreactive response, however, can be beneficial in the elimination of residual leukemia through an adoptive immunotherapy mechanism known as the graft- versus-leukemia (GvL effect). Allogeneic BMT and the use of Alternative Donors: In most instances, the ideal bone marrow donor is the HLA-identical sibling. Alternative donors include the HLA-phenotypically matched unrelated donor (MUD), a partially mismatched related donor (PMRD) or a cord blood donor (CBD), who can be a phenotypically matched or mismatched related or unrelated donor (1). Graft engineering, T cell depletion, and graft-host interactions: Initial attempts to use non-manipulated marrow from MUDs and PMRDs have resulted in severe or fatal GvHD (24,25). This stimulated the development of methods to remove the suspected mediators of GvHD (T lymphocytes) from the marrow ex vivo prior to infusion (26). Results from transplants in which patients received marrow that was highly depleted of T cells (pan-T cell depletion) were initially promising, in that GvHD was significantly reduced; however, this was accompanied by an increase in graft failure (27,28), suggesting that donor T cells may eliminate the ability of residual recipient T cells to reject the graft.

Animal studies of PMRD transplants have indicated that both CD4 and CD8-positive cells are capable of mediating lethal GvHD (29). Initial human studies have therefore used ex vivo pan-T cell depletion to engineer these grafts. This has either been achieved by agglutination with soybean lectin and rosetting the residual T cells with sheep red blood cells, or by use of T cell-directed MAbs, e.g. anti-CD2, CD3, CD5, in combination with panning or complement to eliminate antibody-sensitized cells (26). In a study comparing 470 PMRD reduced the risk of acute GvHD, but increased the risk of graft failure, and there was no overall improvement in leukemia-free survival (30). Therefore, aggressive ex vivo pan- TCD was felt not to be optimal in facilitating PMRD BMT, and subsequent studies have explored the use of a modified pan-T cell depletion that leaves more T cells in the graft. Another option is the use of a more selective or targeted type of TCD often combined with post-transplant immune suppression (11-13).

When T cell depletion (TCD) has been used in matched sibling transplantation, a further concern has been an increase in disease relapse seen particularly in patients with CML (33). This apparent disruption in the graft-versus- leukemia (GvL) effect has discouraged investigators from using TCD other than when MHC-nonidentical grafts are used. We have, however, shown that the use of sequential immunomodulation of the patient and T cell depletion of up to 3Ag PMRD grafts can result in stable and sustained engraftment in >95% of recipients with a low incidence of acute and chronic GvHD (32). Relapse rates in high-risk patients (long-standing recurrent disease or relapse at the time of BMT) can be as high as 70% (2). This indicates that even though it is possible to cross major histocompatibility barriers with successful engraftment and a low incidence of GvHD, further improvement in disease-free survival will depend on the antileukemic effectiveness of the transplant. While this might be accomplished by performing the transplant earlier in the disease course, many patients will not be referred for allogenic BMT until they have demonstrated resistance to conventional-dose therap. Thus, enhancement of the GvL effect may be an essential component of the curative potential of allogeneic BMT.

Biology of the GvL Effect: The GvL reaction is through to be most effective in chronic phase CML (34,35), although there is also evidence for a GvL effect in the acute leukemias (36). It is generally thought that T lymphocytes recognize and eliminate residual leukemia through both MHC restricted and non- restricted pathways (37). Targets for GvL may include minor and/or major mismatched histocompatibility antigens and/or leukemia-specific antigens (38,39). Every allogeneic BMT patient potentially could benefit from the alloreactive response, although the extent of this benefit varies depending on whether the leukemia expresses allogeneic antigens to a degree that triggers recognition and killing. It is known that patients who suffer from acute and chronic GvHD post- BMT often have a reduced rate of leukemic relapse (3,4), possibly due to more intense alloreactivity against residual host-derived leukemic cells. Many investigators have shown evidence that GvHD and GvL effects can be separated to some degree (5,7,40,41), although a system for engineering a GvL effect in total absence of GvHD has not been reliably demonstrated.

T cell recognition of leukemia-associated antigens is also through to be a potentially important means by which immunocompetent cells may recognize and eliminate residual leukemia. It is known that leukemia-reactive clones can be generated (15). Specific targets for leukemia-reactive clones remain the topic of intense investigation, and some potential leukemia-associated antigens have been identified (3,16-19) and are discussed below. The ability to identify and stimulate a GvL effect via either or both of these mechanisms may be of therapeutic importance in reducing the risk of relapse in patients who have received TCD grafts. γδ+ T lymphocytes: Five to ten percent of T cells in normal peripheral blood bear the yδ receptor (42), although this number may be slightly higher in Asians and Blacks. Recent observations suggest that γδ+ T cells play a substantially different role in the immune system than that of β+ T cells. One of the most obvious differences is that most γδ+ T cells usually do not co-express CD4 or CD8, and therefore may develop normally in the absence of MHC class II molecules (43) since positive selection may not be required. Similarly, it is difficult to elicit a response of γδ+ T cells against allogeneic MHC class I or II antigens, and when it has been possible to obtain γδ+ T cell clones against peptide antigens, recognition of these peptides is usually not restricted by classical MHC molecules (44). In addition, γδ+ T cells tend to recognize intact rather than processed polypeptide (44). While the requirements for activation of human γδ+ T cells are still poorly understood, it is clear that they are different from those of β+ T cells, yδ T cells do not require presentation of antigens in the context of the MHC Class I or Class II molecules for activation (45), however, they probably require CD28-mediated co-stimulation, and, following activation, show autocrine IL-2 production (46). They can also be activated by anti-CD2 antibodies (47). γδ+ T cells which express CD25 have also been shown to adhere to fibronectin-coated plates via the VLA-4 receptor with subsequent expansion, and cross linking of VLA-4 and VLA-5 receptors result in co-stimulated expansion induced by an anti pan-δ monoclonal antibody (48). Recent evidence has also suggested that certain subtypes of γδ+ T cells, predominantly the γδ+ CD8αα+ homodimer population, may be resistant to Cyclosporin A (49).

Potential role of TCR-γδ+ T lymphocytes in allogenic BMT: While activation mechanisms for γδ+ T cells are just being elucidated, even less is known about the role of these cells in graft-host interactions. Ellison (50) reported an increase in peripheral γδ+ T cells in murine studies of acute GvHD following allogeneic non-TCD BMT (50). In that study, depletion of γδ+ T cells resulted in a significant decrease in GvHD-related mortality. Blazar (51) also has shown that murine γδ+ T cells can play a role in rejection, alloengraftment, and GvHD through recognition of the "nonclassical" MHC class lb antigens. Studies in humans have to this point been in conflict with murine studies.

Norton (8) did no find γδ+ T cells to be effectors of epidermal damage in cutaneous GvHD. Viale (9) did note an increase in the ratio of Vδ1 ;Vδ2 cells in patients with acute GvHD but the significance of this finding remained undetermined. Tsuji (11) showed that although γδ+ T cells cannot produce GvHD on their own, host γδ+ T cells were recruited into donor αβ+ lesions where they were activated and induced to proliferate. Transitory increases in the ratio of CD4^~CD8^~ γδ+ T cells have been reported during the first four weeks post-BMT in patients treated by GM-CSF, but the cells return to normal levels within eight weeks post-BMT (10). In addition, increased γδ+ T cells have been found in one (study to be associated with viral and fungal infections during the first year following TCD BMT in patients receiving either PMRD or MUD grafts (12). In the same study, increases in γδ+ T cells were not found to be associated with GvHD. The potential for a possible anti-tumor role for γδ+ T cells was established by Esslin (13), who noted that in vitro activated peripheral blood γδ+ T cells posses cytolytic activity to selected human tumor cell lines when compared to similarly activated αβ+ T cells. This reactivity was not MHC restricted, but was dependent on interaction with LFA-1b/ICAM1 rather than the Yδ receptor. These cells predominantly expressed the Vγ9/Vδ2 form of the T cell receptor. Proliferate responses of both αβ+ and γδ+ T cells, however, were inhibited by MAbs to anti- HLA-A, -B, and -C. These findings suggest that γδ+ T cells activated through the TCR have an advantage in non-MHC restricted cytolysis which may correlate with a GvL response. It is known that γδ+ T cells respond to heat shock proteins (16- 18), some of which may be expressed by lymphomas. Human alloreactive γδ+ T cells have also been generated which recognize TCT.1 (Blast-1/CD48), an antigen broadly distributed on hematopoietic cells (52). These γδ+ T cells preferentially expressed the Vγ3/Vδ1 form of the T cell receptor. Vδ1+ cell activation has also been reported in response to EBV-transformed B cells (14,53), EBV-infected Burkitt lymphoma cells (53), and Daudi lymphoma cells (54). In addition, one recent report has shown cytotoxic anti-leukemic activity in a patient against B cell ALL by γδ+ T cells expressing the Vδ1 form of the T cell receptor (19).

We have been able to expand in vitro donor-derived γδ T cells which have a striking resemblance to those seen in the patients described above. Donor mononuclear cells were depleted of CD4+/CD8+ T cells, and expanded on a combination of immobilized pan-δ monoclonal antibody and irradiated recipient B cell leukemia. After initial culture and re-stimulation, the cultures expanded rapidly and contained almost exclusively Vδ1+ γδ+ T cells which expressed CD3, CD25, and CD69, but were CD4- and CD8- which are cytolytic to recipient leukemia and K562 cells but are minimally cytolytic to self MNC and third party leukemia. These observations suggest that donor-derived γδ+ T cells can be generated in vitro, thus providing a potential mechanism for cellular immunotherapy of leukemia.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 shows the expansion of donor γδ+ T cells in culture. Fig. 2 shows the phenotypic analysis of proliferating γδ+ T cells from cultures on pan-δ MAb with blasts.

Fig. 3 shows the phenotypic analysis of proliferating γδ+ T cells from cultures on pan-δ MAb without blasts.

Fig. 4 shows the phenotype of γδ+ T cells from Patient #1. Fig. 5 shows the flow cytometric binding assay depicting the binding of activated donor γδ+ T cells to recipient leukemic CD19+ blasts. Fig. 6 shows the cytotoxicity of donor γδ+ T cells. Fig. 7 shows the cytotoxicity of expanded γδ+ T cells against various cell lines.

Fig. 8 shows the cytotoxic effects of expanded γδ+ T cells against other cell lines.

Fig. 9 shows the mRNA and surface expression of Vδ subtypes. DESCRIPTION OF THE PREFERRED EMBODIMENTS EXPERIMENTAL PROTOCOLS

Donor/recipient pairs: Three patients who presented for BMT with relapsed acute lymphoblastic leukemia or induction failure and their HLA-partially mismatched related donors were enrolled in this study.

Cell preparation: For recipients, sufficient blood was drawn to obtain a minimum of 2.5 x 10⁷ leukemic cells, but less than 50ml, prior to the start of pre-BMT conditioning therapy. Leukemic cells from the recipient were separated from normal mononuclear cells (MNC) using density gradient centrifugation on Percoll using 30-40% gradient. If necessary, further purification was accomplished by immunomagnetic o depletion of normal T and NK cells. Purity of normal and blast monolayers were evaluated by flow cytometry using MAbs previously found to be diagnostic for the patient's leukemia. The cells were then cryopreserved at a concentration of 20 x 10⁶/ml in AIM-5 medium (Gibco) with 15% fetal bovine serum (Gibco) and 10% DMSO and stored in liquid nitrogen until donor selection was complete. Up to 50 ml of peripheral blood was then obtained from the corresponding partially mismatched related donor. These donor- derived γδ+ T cells were purified in the MNC layer by negative selection using CD4+ and CD8+ immunomagnetic microspheres (Dynal) at a ratio of 5 microspheresxell. Removal of CD4+ and CD8+ cells from peripheral blood effectively depleted >95% of αβ+ T cells. The number of γδ+ T ceils in the preparation and the effectiveness of the αβ+ T cell depletion was monitored by flow cytometry as described below using fluorochrome- conjugated antibodies to TCR-αβ, TCR-γδ, CD4, CD8, and CD3 (Becton-Dickinson Immunocytometry Systems-BDIS; San Jose, CA).

Culture and activation of γδ+ T cells: Cytotoxic γδ+ T cells were generated from donor-recipient pairs as follows: Tissue culture-treated 24 well plates were coated with 10μg TCR-δ1 pan-δ monoclonal antibody (Endogen; Woburn, MA) in 300μg PBS for 24h at 4°C to facilitate initial activation and expansion of γδ+ T cells as described by Esslin (13). Irradiated (50Gy) primary leukemic blasts that were obtained and cryopreserved prior to BMT were thawed, washed x 3, and re-suspended in AIM-5 Media with 15% FBS and 25IU of IL-2 at a concentration of 1.0 x 10⁶ cells/ml. Aliquots of 1 ml of this suspension were plated on the coated wells. Following immunomagnetic depletion of CD4+ and CD8+ cells as described above, remaining donor-derived MNC were adjusted to a concentration of 1.0 x 10⁶ cells/ml, and aliquots of 1 ml were added to the previously plated recipient blasts. Control wells consisted of CD4+CD8+ depleted MNC plated on TCR-δl monoclonal antibody in the absence of blasts or blasts in the absence of monoclonal antibody. The cultures were examined daily for characteristic morphology of proliferating clusters. Media was refreshed twice weekly or as necessary dependent on the robustness of proliferation as determined by microscopic examination and the phenol red pH indicator in the media. After two weeks in culture, cells were photographed, and subcultured 1 :2 or 1 :4 as necessary onto a freshly coated plate. The γδ+ T cell + blast wells were re-stimulated with freshly thawed blasts at the same concentration used previously and assayed at this time and weekly thereafter for phenotype, Vδ subtype, and absolute cell number. At week four, fold expansion was calculated and harvesting was begun for phenotypic, molecular, and functional assays described below and for cryopreservation and storage as described above for future study. These assays were performed at 4 - 6 weeks of culture. The concentration of γδ+ T cells measured on a biweekly basis determined the degree of γδ+ T cell stimulation^" for each culture condition. When necessary, proliferating cells were transferred onto pan-δ MAb-coated tissue cultured flasks (Becton Dickinson) and cultures were maintained for up to twelve weeks, at which time no further proliferation was observed.

Flow cytometry: Expanded/activated γδ+ T cells were analyzed by four color flow cytometry for expression of CD45, CD3, CD4, CD8, CD19, CD56, CD25, HLA-DR, CD69 (Becton Dickinson Immunocytometry Systems; San Jose, CA-BDIS), and Vδ1 (Endogen, Woburn, MA), TCR-γδ, CD57, CD94, and Vδ1-Vδ3 (Coulter Immunotech; Miami, FL) using monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), or allophycocyanan (APC). Recipient primary B cell leukemias were analyzed for expression of CD19, CD10, CD45, CD7, CD20, CD23, slgGκ, slgGλ, HI_A-ABC, and HLA-DR (all from BDIS). At least 50,000 ungated events were collected in a list mode file and cell subpopulations in the lymphocyte CD45/side scatter gate and CD3/side scatter gate are quantitated and expressed as a percentage of the total lymphocyte population. Analysis was performed on a FACS Calibur flow cytometer using CellQuest software (BDIS).

Flow cytometric binding assays. Binding of donor γδ+ T cells to specific targets was examined by flow cytometry. Donor γδ+ T cells were incubated in AIM-5 Media with 15% FBS for 30 minutes at 37°C, centrifuged, and resuspended in phosphate-buffered saline. The cell suspension was labeled with one MAb specific for the leukemia but not expressed on γδ+ T cells (CD19) and anti-TCR γδ, which is not expressed on the leukemia. The cell preparation was incubated at 4°C for 30 min, washed x 3, and analyzed by flow cytometry as detailed above. Clusters which were positive for both CD19 and γδ were then examined for forward (FSC) and side scatter (SSC) to determine if the represented multi-cell clusters. Dual-positive cells with increased FSC and SSC were scored as bound blast/γδ+ T cell clusters. Controls consisted of cultures of resting and activated donor γδ+ T cells co-cultured K562 cells. K562 cells are autofluorescent, so labeling with a flurochrome was unnecessary. Resting γδ+ T cells do not bind K562 while activated γδ+ T cells do.

Cytotoxicity assays: Third-party mononuclear cells, K562 erythroleukemia cells, and recipient primary leukemia were used as targets. Aliquots of target cells were labeled overnight with 3,3'-dioctadecyloxacarbocyanine (DiOC,₈) (Molecular Probes, Eugene, OR). The cells were then washed in phosphate buffered saline (PBS) and resuspended in RPMI-1640 with 10% fetal bovine serum (FBS) at a concentration of 2 x 10* cells/ml. Control MNC and expanded γδ+ T cells were suspended in RPMI-1640 and diluted to yield E:T ratios of 40:1-2.5:1 and added to the target cells. Aliquots of 130μl counterstaining solution consisting of propidium iodine (PI) and PBS (Molecular Probes) were then added to the cell mixtures. The tubes were pelleted by centhfugation at 1000 x g for 30 sec and then incubated for 4 hours. Following incubation, the tubes were acquired in a FACS Calibur flow cytometer (BDIS) and analyzed for green fluorescence (DiOC_1β-560nm) and red fluorescence (Pl-630nm). Analysis on a two parameter histogram allows separation of live target cells (DiOC,_β+PI-) and membrane-compromised targets are (DiOC_1β+PI+) from which % cytotoxicity was calculated. γδ T cell Receptor Characterization: The clonal heterogeneity of γδ+ T cells determined by flow cytometry was further evaluated using molecular approaches to assess γδ TCR variable gene expression using peripheral blood mononuclear cells (PBMC) collected from the BMT donors and the expanded γδ+ T cells from derived from culture on the pan-δ MAb and co-culture with the recipient ALL. Total RNA was extracted from MNC or cultured cells by the acid-phenol guanidinium thiocyanate method (55) and reverse transcribed according to the GeneAmp RNA PCR protocol (Perkin-Elmer Cetus, Norwalk, CT). The cDNA product served as template for PCR amplifications utilizing γδ TCR gene family- specific primers according to established methods (56). PCR amplification products were analyzed by agarose gel electrophoresis in order to determine the number and identity of γδ TCR V gene families expressed in each sample. This analysis was facilitated by DNA blot hybridization with corresponding TCR Cγ - or Cδ -horseradish peroxidase (HRP) conjugated oligonucleotide probes followed by chemiluminescent detection (23). Amplified products were resolved on 4% sequencing gels and detected, due to the incorporation of fluorescent primers during amplification, using the Hitachi FMBIO-100 Fluorescent Imager or the ABI 377 (Perkin-Elmer) automated sequencer using Genescan™ software. This method (known as TCR spectratyping) provides a more refined assessment of γδ TCR clonal diversity in the specimens. RESULTS

Immobilized pan-δ MAb alone and with and leukemic blasts stimulate γδ+ T cells. As shown in Figure 1 , γδ+ T cells strongly proliferated in response to immobilized pan-δ MAb alone and a combination of immobilized pan-δ MAb and blasts. Leukemic blasts alone did not support sustained proliferation of γδ+ T cells. It should be noted, however, that in one experiment γδ+ T cell proliferation occurred later in the culture than in the other two experiments. Immunophenotypic analysis of proliferating γδ+ T cell cultures. Phenotypic analysis revealed that proliferating γδ+ T cells from cultures on pan-δ MAb with blasts preferentially expressed Vδ1 (Figure 2) while γδ+ T cells proliferating on pan-δ MAb without blasts preferentially expressed Vδ2 (Figure 3). The γδ+ T cell cultures were predominantly CD3+CD4-CD8- and expressed activation-associated antigens CD69, CD25, and HLA-DR regardless of culture conditions (Figure 4). Functional analysis ofyδ+ T cell cultures. Cultured donor-derived γδ+ T cells from both culture methods were tested for their ability to bind and to lyse primary leukemia from the corresponding BMT recipient. Figure 5 shows that indeed donor γδ+ T cells will bind recipient leukemia. Donor γδ+ T cells were highly cytotoxic to recipient leukemia as well as the NK sensitive target cell line K562 (Figure 6). In one experiment, mild nonspecific cytotoxicity was seen against third party MNC. Different lytic profiles were seen which correlated with culture method and predominant Vδ gene usage (Figures 7 & 8). Vδ1+ cells cultured on immobilized pan-δ MAb and recipient blasts lysed primary ALL from the recipient and K562 cells as well as lymphoid cell lines, but had essentially no activity against myeloid cell lines. In contrast, Vδ2 clones from cultures expanded on pan- δ MAb alone showed cytotoxic activity against all targets. TCR repertoire analysis of γδ+ T cells. Polyclonal γδ+ T cells from the healthy BMT donors expressed mRNA predominantly for Vδ2 followed by Vδ1 and the Vδ3 (Figure 9). Occasionally mRNA for Vδ4 and Vδ5 was seen. Examination of the Vδ repertoire of γδ cells cultured on pan-δ MAb alone was essentially unchanged from the peripheral blood Vδ repertoire. In contrast, γδ+ T cells cultured on pan-δ MAb and blasts showed preferential expression of Vδ1 , followed by Vδ2 and Vδ3. High resolution analysis of these PCR products revealed. It will be apparent to those of ordinary skill in the art that many modifications and substitutions can be made without departing from the spirit and the scope of the present invention.

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Claims

IN THE CLAIMS:

1. A cell line comprising substantially purified γδ+ T lymphocytes.

2. The cell line of claim 1 , wherein said γδ+ T lymphocytes preferentially express the Vδ1 phenotype.

3. The cell line of claim 1 , wherein said γδ+ T lymphocytes preferentially express the Vδ2 phenotype.

4. A method of treating leukemia comprising the steps of: administering to a leukemic patient a suitable amount of substantially purified γδ+ T cells; and monitoring said patient for the presence of leukemia.

5. The method in accordance with claim 4, wherein said γδ+ T lymphocytes preferentially express the Vδ1 phenotype.

6. The method in accordance with claim 4, wherein said γδ+ T lymphocytes preferentially express the Vδ2 phenotype.

7. A method of preparing substantially purified γδ+ T cells comprising the steps of: incubating donor γδ+ T cells with irradiated recipient leukemia to allow activation and expansion of γδ+ T cells; and harvesting said γδ+ T cells.