AU771710B2 - In vitro activated gamma delta lymphocytes - Google Patents

Description

WO 00/44893 PCT/USOO/01867 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 Indeed, relapse rates in high-risk patients (long-standing recurrent disease or relapse at the time of BMT) can be as high as 70% Therefore, further improvement in diseasefree 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 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 y8+ T cells may not be important primary effectors of GvHD few have addressed the GvL potential of y+ T cells. Esslin (13) noted that in vitro activated y6+ T cells can mediate broadly-based non-MHC restricted cytolytic activity to selected human tumor cell lines. Others have shown that y6+ 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 y+ T cells expressing the V51 form of the T cell receptor Taken together, these findings support a potential antileukemic role for y8+ T cells.

Published data describing a series of 10 leukemia patients who developed an increased proportion of circulating CD3+CD4-CD8-V61+y8+ T cells between WO n/448o3 PCT/USOO/01867 2 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 y8+ T cells In addition, it has been recently shown that enrichment of the graft with yS+ T cells may have contributed to the later development of increased y3+ T cells Regardless of the TCD protocol used, however, patients who developed increased y8+ 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 The same alloreactive response, however, can be beneficial in the elimination of residual leukemia through an adoptive immunotherapy mechanism known as the graftversus-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 Graft engineering, T cell depletion, and graft-host interactions: Initial attempts to use non-manipulated marrow from MUDs and PMRDs have resulted Wn nnA/44O PCT/US00/01867 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 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 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 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 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 This apparent disruption in the graft-versusleukemia (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 Relapse rates in high-risk patients (long-standing recurrent disease or relapse at the time of BMT) can be as high as 70% 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 wO 0nA4O3 PCT/US00oo/0o1867 UJCU fvlf s 4 PCT/USOO/ 167 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 It is generally thought that T lymphocytes recognize and eliminate residual leukemia through both MHC restricted and nonrestricted pathways 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 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 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.

y6+ T lymphocytes: Five to ten percent of T cells in normal peripheral blood bear the y6 receptor although this number may be slightly higher in Asians and Blacks. Recent observations suggest that y6+ T cells play a WO 00/44893 PCTIUSOO/01867 substantially different role in the immune system than that of ap+ T cells. One of the most obvious differences is that most y6+ 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 yi+ T cells against allogeneic MHC class I or II antigens, and when it has been possible to obtain y6+ T cell clones against peptide antigens, recognition of these peptides is usually not restricted by classical MHC molecules In addition, y5+ T cells tend to recognize intact rather than processed polypeptide (44).

While the requirements for activation of human y6+ T cells are still poorly understood, it is clear that they are different from those of ap+ T cells. y6 T cells do not require presentation of antigens in the context of the MHC Class I or Class II molecules for activation 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 y6+ 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 receptors result in co-stimulated expansion induced by an anti monoclonal antibody Recent evidence has also suggested that certain subtypes of y6+ T cells, predominantly the y6+ CD8aa+ homodimer population, may be resistant to Cyclosporin A (49).

Potential role of TCR-y6+ T lymphocytes in allogenic BMT: While activation mechanisms for y6+ 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 y6+ T cells in murine studies of acute GvHD following allogeneic non-TCD BMT In that study, depletion of y6+ T cells resulted in a significant decrease in GvHD-related mortality. Blazar (51) also has shown that murine y6+ T cells can play a role in rejection, alloengraftment, and GvHD through recognition of the "nonclassical" MHC class Ib antigens.

Studies in humans have to this point been in conflict with murine studies.

Norton did no find y6+ T cells to be effectors of epidermal damage in cutaneous GvHD. Viale did note an increase in the ratio of V51;V52 cells in PCTIUSOO/01867 lll I JIA A€>\1 VW UU/qt07 v 6- 6 patients with acute GvHD but the significance of this finding remained undetermined. Tsuji (11) showed that although y6+ T cells cannot produce GvHD on their own, host yi+ T cells were recruited into donor ap+ lesions where they were activated and induced to proliferate. Transitory increases in the ratio of CD4-CD8- yV+ 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 In addition, increased yS+ 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 In the same study, increases in y6+ T cells were not found to be associated with GvHD.

The potential for a possible anti-tumor role for y6+ T cells was established by Esslin who noted that in vitro activated peripheral blood y6+ T cells posses cytolytic activity to selected human tumor cell lines when compared to similarly activated a T cells. This reactivity was not MHC restricted, but was dependent on interaction with LFA-lb/ICAM1 rather than the y6 receptor. These cells predominantly expressed the Vy9N62 form of the T cell receptor. Proliferate responses of both ap+ and y6+ T cells, however, were inhibited by MAbs to anti- HLA-A, and These findings suggest that y5+ 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 T cells respond to heat shock proteins (16- 18), some of which may be expressed by lymphomas. Human alloreactive y5+ T cells have also been generated which recognize TCT.1 (Blast-1/CD48), an antigen broadly distributed on hematopoietic cells These y6+ T cells preferentially expressed the Vy3/V1 form of the T cell receptor. V61+ cell activation has also been reported in response to EBV-transformed B cells (14,53), EBV-infected Burkitt lymphoma cells and Daudi lymphoma cells In addition, one recent report has shown cytotoxic anti-leukemic activity in a patient against B cell ALL by y6+ T cells expressing the V51 form of the T cell receptor (19).

We have been able to expand in vitro donor-derived y8 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-8 monoclonal antibody and irradiated recipient B wn nn/44o PCT/US00/01867 cell leukemia. After initial culture and re-stimulation, the cultures expanded rapidly and contained almost exclusively V81+ y6+ T cells which expressed CD3, 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 yS+ 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 y8+ T cells in culture.

Fig. 2 shows the phenotypic analysis of proliferating y8+ T cells from cultures on pan-8 MAb with blasts.

Fig. 3 shows the phenotypic analysis of proliferating y6+ T cells from cultures on pan-5 MAb without blasts.

Fig. 4 shows the phenotype of y5+ T cells from Patient #1.

Fig. 5 shows the flow cytometric binding assay depicting the binding of activated donor y8+ T cells to recipient leukemic CD19+ blasts.

Fig. 6 shows the cytotoxicity of donor y8+ T cells.

Fig. 7 shows the cytotoxicity of expanded y6+ T cells against various cell lines.

Fig. 8 shows the cytotoxic effects of expanded y8+ T cells against other cell lines.

Fig. 9 shows the mRNA and surface expression of VS 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 10' 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 WO nn/44893 PCT/US00/01867 8 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 10/ml in 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 donorderived y8+ T cells were purified in the MNC layer by negative selection using CD4+ and CD8+ immunomagnetic microspheres (Dynal) at a ratio of 5 microspheres:cell. Removal of CD4+ and CD8+ cells from peripheral blood effectively depleted >95% of ap+ T cells.

The number of y8+ T cells in the preparation and the effectiveness of the ap+ T cell depletion was monitored by flow cytometry as described below using fluorochromeconjugated antibodies to TCR-oa, TCR-yS, CD4, CD8, and CD3 (Becton-Dickinson Immunocytometry Systems-BDIS; San Jose, CA).

Culture and activation of y5+ T cells: Cytotoxic y8+ T cells were generated from donor-recipient pairs as follows: Tissue culture-treated 24 well plates were coated with TCR-81 pan-8 monoclonal antibody (Endogen; Woburn, MA) in 300pg PBS for 24h at 4oC to facilitate initial activation and expansion of y8+ T cells as described by Esslin 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 251U of IL-2 at a concentration of 1.0 x 10" cells/ml. Aliquots of 1ml 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/mi, and aliquots of 1 ml were added to the previously plated recipient blasts. Control wells consisted of CD4+CD8+ depleted MNC plated on TCR-81 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 y8+ 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, V8 subtype, and absolute cell number. At week four, fold expansion was calculated and harvesting was W 00nn/44R3 PCT/USOO/01867 9 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 y8+ T cells measured on a biweekly basis determined the degree of yS+ T cell stimulation for each culture condition.

When necessary, proliferating cells were transferred onto pan-8 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 y 8 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 V81 (Endogen, Woburn, MA), TCR-y8, CD57, CD94, and V81-V83 (Coulter Immunotech; Miami, FL) using monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC), phycoerythrin peridinin chlorophyll protein (PerCP), or allophycocyanan (APC). Recipient primary B cell leukemias were analyzed for expression of CD19, CD10, CD45, CD7, CD20, CD23, sigGic, slgGX, HLA-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 y8+ T cells to specific targets was examined by flow cytometry. Donor y8+ T cells were incubated in 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 y8+ T cells (CD19) and anti-TCR y8, 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 yS 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 blastly8+ T cell clusters. Controls consisted of cultures of resting and activated donor y8+ T cells co-cultured K562 cells.

WO 00/44893 PCT/US00/01867 K562 cells are autofluorescent, so labeling with a flurochrome was unnecessary. Resting y8+ T cells do not bind K562 while activated y8+ 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,,) (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 4 cells/ml. Control MNC and expanded y8+ 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 130 1 counterstaining solution consisting of propidium iodine (PI) and PBS (Molecular Probes) were then added to the cell mixtures. The tubes were pelleted by centrifugation 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,,-560nm) and red fluorescence (PI-630nm).

Analysis on a two parameter histogram allows separation of live target cells (DiOC,,+PI-) and membrane-compromised targets are (DiOC,,+PI+) from which cytotoxicity was calculated.

yS T cell Receptor Characterization: The clonal heterogeneity of y+ T cells determined by flow cytometry was further evaluated using molecular approaches to assess y8 TCR variable gene expression using peripheral blood mononuclear cells (PBMC) collected from the BMT donors and the expanded y6+ T cells from derived from culture on the pan-8 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 y6 TCR gene familyspecific primers according to established methods PCR amplification products were analyzed by agarose gel electrophoresis in order to determine the number and identity of y6 TCR V gene families expressed in each sample. This analysis was facilitated by DNA blot hybridization with corresponding TCR Cy or CS -horseradish peroxidase (HRP) conjugated oligonucleotide probes followed by wn n/4489 PCT/US00/01867 11 chemiluminescent detection 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 yS TCR clonal diversity in the specimens.

RESULTS

Immobilized pan-8 MAb alone and with and leukemic blasts stimulate y8+ T cells.

As shown in Figure 1, yS+ T cells strongly proliferated in response to immobilized pan-8 MAb alone and a combination of immobilized pan-8 MAb and blasts.

Leukemic blasts alone did not support sustained proliferation of yS+ T cells. It should be noted, however, that in one experiment y8+ T cell proliferation occurred later in the culture than in the other two experiments.

Immunophenotypic analysis of proliferating yS+ T cell cultures. Phenotypic analysis revealed that proliferating yS+ T cells from cultures on pan-8 MAb with blasts preferentially expressed V81 (Figure 2) while y6+ T cells proliferating on pan-8 MAb without blasts preferentially expressed V62 (Figure The yS+ 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 ofy8+ T cell cultures. Cultured donor-derived y8+ 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 yS+ T cells will bind recipient leukemia. Donor yS+ T cells were highly cytotoxic to recipient leukemia as well as the NK sensitive target cell line K562 (Figure In one experiment, mild nonspecific cytotoxicity was seen against third party MNC. Different lytic profiles were seen which correlated with culture method and predominant VS gene usage (Figures 7 V81+ cells cultured on immobilized pan-8 MAb and recipient blasts lysed primary ALL from the recipient and K562 cells as well as lymphoid cell lines, but had essentially no activity wn 0n/dd93 PCT/US00/01867 12 against myeloid cell lines. In contrast, V52 clones from cultures expanded on pan- 8 MAb alone showed cytotoxic activity against all targets: TCR repertoire analysis ofy+ T cells. Polyclonal y8+ T cells from the healthy BMT donors expressed mRNA predominantly for V82 followed by V81 and the V83 (Figure Occasionally mRNA for V84 and V55 was seen. Examination of the repertoire of y8 cells cultured on pan-S MAb alone was essentially unchanged from the peripheral blood VS repertoire. In contrast, y8+ T cells cultured on pan-8 MAb and blasts showed preferential expression of V61, followed by V52 and V63.

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 (11)

1. A method of treating a patient with acute lymphoblastic leukemia comprising the steps of: obtaining a donor material comprising bone marrow or peripheral blood collected from a donor; depleting said donor material in vitro of ap+ T cells, thereby rendering the donor material rich in y 6 T cells; activating said y8+ T cells in vitro by treating said donor material with a media comprising leukemia blast cells extracted from the patient and irradiated ex vivo, alone or in combination with a pan-8 monoclonal antibody, so as to render said y6+ T cells cytolytic to the patient's leukemia cells but minimally cytotoxic to the patient's other cells; and administering an effective amount of said activated y8+ T cells to said leukemia patient.

2. The method in accordance with claim 1, wherein said ap donor material is obtained by negative selection.

3. The method in accordance with claim 2, wherein said negative selection is performed using immunomagnetic microspheres associated with either CD4+ and 20 CD8+ or an anti-TCR 4ap monoclonal antibody.

4. The method in accordance with claim 1, wherein said activated y8 T cells predominantly express the V82 phenotype.

5. The method in accordance with claim 1, wherein said activated y8 T cells predominantly express the V82 phenotype. *o 25 6. The method in accordance with claim 1, wherein said 4ap T cells are depleted by depleting the donor material of CD4+ and CD8+ T cells.

7. The method in accordance with claim 6, wherein said pan-s antibody comprises an anti-TCR-pan-6 antibody.

8. The method in accordance with claim 1, wherein said activation step comprises treatment with leukemia blast cells, an anti-TCR-pan-6 antibody, and interleukin-2.

9. The method in accordance with claim 6, wherein said media consists essentially of irradiated leukemia blast cells extracted from said patient, an anti- TCR- pan-8 antibody, and interleukin-2. The method in accordance with claim 1, wherein said donor material is derived from an HLA-partially mismatched related donor.

11. The method in accordance with claim 1, wherein said donor material is derived from an HLA-identical sibling.

12. The method in accordance with claim 1, wherein donor material is derived from an HLA-phenotypically matched unrelated donor.

13. The method in accordance with claim 1, wherein donor material is derived from a cord blood donor. 20 Dated this 23 rd January 2004 PALMETTO HEALTH ALLIANCE d/b/al Palmetto By their Patent Attorneys o COLLISON CO