Patent Application: US-87939801-A

Abstract:
patients who develop increased numbers of γδ + cytotoxic t lymphocytes 2 - 6 months after allogeneic bone marrow transplantation are less likely to relapse than those who do not . the γδ + t cells isolated from blood of patients with increased γδ + t cells are cd3 + cd4 − cd8 − cd57 +, cytolytic to k562 cells , and express the vδ1 t cell receptor phenotype . similar γδ + t cells can be generated in vitro by culture of donor mononuclear cells which are enriched for γδ + t cells by immunomagnetic depletion of depleted of cd4 + and cd8 + cells . this γδ - 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 leulemia cell lines . these observations suggest that donor - derived cytotoxic γδ + t cells can be generated in vitro , and may provide therapeutic potential for prevention of disease relapse .

Description:
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 × 10 7 leukemic cells , but less than 50 ml , 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 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 &# 39 ; s leukemia . the cells were then cryopreserved at a concentration of 20 × 10 6 / 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 microspheres : cell . removal of cd4 + and cd8 + cells from peripheral blood effectively depleted & gt ; 95 % of αβ + t cells . the number of γδ + t cells 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 , calif .). 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 10 μg tcr - δ1 pan - δ monoclonal antibody ( endogen ; woburn , mass .) in 300 μg pbs for 24 h at 4 ° c . to facilitate initial activation and expansion of γδ + t cells as described by esslin ( 13 ). irradiated ( 50 gy ) primary leukemic blasts that were obtained and cryopreserved prior to bmt were thawed , washed × 3 , and re - suspended in aim - 5 media with 15 % fbs and 25 iu of il - 2 at a concentration of 1 . 0 × 10 6 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 × 10 6 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 - δ1 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 restimulated 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 , mass . ), tcr - γδ , cd57 , cd94 , and vδ1 - vδ3 ( coulter immunotech ; miami , fla .) 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 , siggκ , siggλ , 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 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 × 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 18 ) ( molecular probes , eugene , oreg .). 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 × 10 4 cells / mi . 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 130 μl 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 × 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 18 - 560 nm ) and red fluorescence ( pi - 630 nm ). analysis on a two parameter histogram allows separation of live target cells ( dioc 18 + pi −) and membrane - compromised targets are ( dioc 18 + 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 , conn .). 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 . immobilized pan - δ mab alone and with and leukemic blasts stimulate γδ + t cells . as shown in fig1 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 ( fig2 ) while γδ + t cells proliferating on pan - δ mab without blasts preferentially expressed vδ2 ( fig3 ). the γδ + t cell cultures were predominantly cd3 + cd4 − cd8 − and expressed activation - associated antigens cd69 , cd25 , and hla - dr regardless of culture conditions ( fig4 ). functional analysis of γδ + 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 . fig5 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 ( fig6 ). 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 ( fig7 & amp ; 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 ( fig9 ). 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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