Patent Application: US-80857407-A

Abstract:
the invention provides a method for determining antigen - specific t cell response in a microarray system consisting of microwells wherein a t - cell preparation is challenged with the antigen and the cytokines thereby released are detected with an immunoassay .

Description:
it has been found that an effective , highly - sensitive determination of antigen - specific t cell response can be performed in a high troughput format , using cultures of as few as 5 × 10 3 to 50 × 10 3 cells dispensed in microwells with 5 to 50 μl capacity . accordingly , the invention provides a method for determining antigen - specific t cell response in a high throughput format , which comprises the following steps : i ) providing a multiwell plate suitable for culturing cells in a volume of from 5 to 50 μl / well ; ii ) coating the wells with anti - cytokine antibodies ; iii ) adding the test antigen to the wells ; iv ) providing a pbmc ( peripheral blood mononuclear cells ) or lymphocyte culture and dispensing it in the wells in amounts of from 5 × 10 3 to 50 × 10 3 pbmcs or lymphocytes per well ; v ) incubating the cells for a time sufficient for antigen - mediated t cell stimulation to occur ; vi ) detecting the presence of the cytokine secreted in the culture medium and immobilized by the anti - cytokine antibody of step ii ), by means of an immunoassay . in a preferred embodiment the assay is carried out in a 384 or 1536 well plate and the pbmcs or the lymphocytes are preferably separated from human blood samples . because of the use of very few cells in comparison to current methods , this is ideal for lymphopenic patients or for small volume samples ( pediatric patients ). in this system , autologous apc are present in the pbmc preparation . monocytes , b cells , dendritic cells expressing hla class ii alleles can present to cd4 lymphocytes either proteins that require processing or peptides that directly bind to class ii molecules . all mononucleated cells expressing hla class i molecules ( t and b lymphocytes , monocytes , dendritic cells , nk cells ) are adequate for direct presentation of class i restricted peptides . for more accurate simulation of class i restricted presentation to cd8 cells , virus infected cells can also be added to the wells , while this is not feasible with mhc - peptide chip arrays . according to the invention , the antigens may include hla class i or class ii restricted peptides , proteins , virus , virus - infected cell , cell - or virus lysate , for example tetanus toxin , ppd ( m . bovis ), cmv pp65 and ie - 1 protein , cmv lysate , t . gondii lysate , lysates of p . carinii , aspergillus sp . and cryptococcus cells , sars coronavirus spike protein peptides , hiv and htlv1 peptides and proteins , selected immunodominant peptides from adenovirus , respiratory syncytial virus and other relevant viruses . cytokines are rapidly synthesized by specific t cells upon antigen recognition . according to the invention , the cytokines preferably are il2 , il4 , il5 , ifng , tnfa and gm - csf . in order to have a cytokine production in detectable amounts , the t cells are generally incubated in the presence of the antigens for a time period of 18 - 24 hrs . in a preferred embodiment of the invention , the cytokines secreted in the culture medium and immobilized by the anti - cytokine antibody are detected by elisa . the latter preferably comprises the following steps : i ) contacting a labeled antibody with the antibody - cytokine complex , to form an antibody - cytokine - labeled antibody complex ; ii ) washing ; iii ) contacting the complex formed in step i ) with a labeled enzyme able to produce a detectable signal upon reaction with the substrate , to form a antibody - cytokine - labeled antibody - labeled enzyme complex ; iv ) washing ; v ) contacting the enzyme in the complex formed in step iii ) with the substrate ; vi ) detecting the signal produced by the enzyme - substrate reaction . preferably the antibody used in step i ) is biotinylated and the labeled enzyme used in step iii ) is alkaline phosphatase conjugated streptavidin or streptavidin conjugated to other convenient enzymes ( e . g . peroxidase , beta - galactosidase ). this assay can be be applied to large scale screening of peptide panels to identify peptides that activate t cells , irrespective of the individual cytokines that are produced . in particular , by using the so called polyelisa test , positive supernatants that contain anyone of the cytokines revealed by the polyelisa test can be detected . this identifies “ cytokine positive wells ”. if supernatants have been saved in replica plates , selected positive samples can be subsequently examined for individual cytokines in miniaturized formats ( microelisa , luminex assays , cytokine arrays ). it was observed that the ratio between positive response in the presence of antigen and negative response ( background in the absence of antigen ) is higher than in conventional proliferation assays and in intracytoplasmic staining . this enhanced differential suggests that cell elisa is more efficient to reveal low responses , in comparison to other current methods . with respect to sensitivity , the ifng produced by a single t cell can be detected by titrating a t cell line into non responding pbmc ( 1 t cell out of 50 , 000 pbmc , corresponding to a frequency of 0 . 002 %). limiting dilution experiments confirmed that one single cell can be identified based on poissonian distribution of the statistically significant positive signals . based on limiting dilution experiments with specific t cell lines or on titrations of pbmc containing specific cd8 t cells defined by tetramer enumeration , ifng produced by one single cell in 24 hours ranges between 0 . 5 and 5 pg . 1 . basic research : epitope mapping , definition of cytokine profiles by microcultured cells ( e . g . cloned cells ) or by selected cell subsets . specificity screening for antigens / peptides derived from pathogens , tumor antigens , autoantigens , alloantigens 2 . clinical investigation : epitope mapping in infected patients , in vaccinated subjects enrolled in vaccine trials , in tumor patients , in transplant recipients , in patients suffering from autoimmune diseases . 3 . diagnostic applications : definition of cellular immunity ( immunocompetence ) specific for a broad range of relevant pathogens . by using minute amounts of cells , the test is particularly suitable for lymphopenic patients and pediatric patients . 4 . diagnostics in developing countries : since the assay is simple , inexpensive , fast and requires little technical training , cellular immune responses to poverty related infections like hiv , malaria , tb , hcv , etc can be easily detected and monitored by using pre - coated plates with pre - dispensed antigens in the field . 5 . industrial applications : high throughput screening of compounds with known or unknown functions to test their interference with cellular immune responses ( immunomodulators ). this may lead to the discovery of new immunosuppressive or immunoenhancing compounds , since screening of thousands of compounds can be easily performed with limited cost and in a short period of time in the 1536w format . 6 . veterinary applications : by using anti human antibody pairs cross reacting with the species under study , or species - specific antibody pairs , it is possible to screen cellular immunity in a variety of animal species , including primates ( also used for basic research ) or farmed animals that may be affected by epidemic diseases . the diagnosis of cellular immunity may provide earlier information than serology in viral diseases , and essential information when antibody responses are not significant ( e . g . mycobacterial infections ). the attached figures and the section “ materials and methods ” illustrate the invention in greater detail . cellelisa , left panel : ifng production by pbmc upon antigen stimulation , 24 hrs , pbmc ( 50 × 10 3 / well in 50 μl ) from donors mdm and mp were cultured in a 384w plate precoated with a - ifng antibodies , in the presence of different recall antigens . after 24 hrs , sups were removed and wells were developed for elisa . pbmc cultures were set up with donor mp as described in fig3 . supernatants were removed at the indicated times and wells were filled with pbs - tween . after 60 hrs , all wells were developed for elisa . it can be seen a slight but gradual increase in background , while ifng induced by antigen stimulation ( tt , ppd , cmv ) reaches a plateau at 36 - 48 hrs . for convenience , an incubation time of 20 - 24 hrs was chosen in subsequent assays . in order to further scale down the culture volumes , we run assays in 1536w plates , that received antigens in 1 μl volume and cells in 10 μl volume ( 10e4 pbmc / well ). the same conditions were used to seed 72 well plates ( terasaki plates ) or 384 well plates with conical shape wells that take up to 20 μl working volume . after culturing and processing the wells as described , the developed substrate pnpp was harvested from four replicate wells from the 1536w plate and read as 40 μl in a 384w plate ( in fact we do not have a 1536 plate reader available at the present time ). the figure shows that excellent results can be achieved also in these conditions , indicating that both cultures and elisa assays can be scaled down to the 1536w format or to the 10 μl volume . comparison of cellelisa with standard methods for evaluation of specific t cell responses : cellelisa vs intracytoplasmic ifn staining vs proliferation . cellelisa and icc were tested after 24 hrs , proliferation after 5 days . results are given as od , % positive cells and kcpm respectively . stimulation indexes for the different assays are shown on the right .. the highest indexes , indicating the highest differential between unstimulated and stimulated pbmc , were obtained by cellelisa . cd4 t cell line specific for cmv pp65 peptide 30 ( 117 - 131 , plkmlnipsinvhhy ) ( containing 100 % specific cells according to icc after & gt ; 6 restimulation cycles with antigen and apc ) was spiked into autologous irradiated ( non responding ) pbmc . six replicate wells were set up for each t cell dilution . results are shown as average value of replicates . background response of irradiated pbmc to pep30 ( 120 od ) was subtracted . it can be seen that 1 cell / well still yields a positive signal , corresponding to detection of 1 specific cell out of 50 , 000 pbmc ( 1 / 50 , 000 = 0 . 002 %). the cd4 t cell line was diluted into autologous irradiated pbmc so to have 1000 , 100 , 10 , 1 cell per well in 16 replicates , plus 50 × 10ˆ3 pbmc per well as apc . positive wells were defined as those giving a signal higher than the mean value of negative background wells ( receiving no t cells )+ 2 sd ( 210 od ). all wells with 10 or & gt ; 10 specific t cells were positive . among the wells with 1 specific t cell , 62 % were positive and 38 % were negative . this matches the expected theoretical frequency of 63 % positive and 37 % negative for a single hit event . pbmc from different donors were cultured with various antigens ( tetanus toxoid , ppd , cmv , c . albicans , asp . niger , p . carinii , pool of cmvpp65 th peptides , pool of cmv pp65 ctl peptides , pha , cmv peptides ( nv9 = a2 , ta9 - cl9 = b7 ), t . gondii ). representative results are shown in 5 a in an array format , with shades corresponding to od ranges ( left panel ). 5 b . two donors were also screened with a panel of overlapping peptides that encompas the immunodominant protein pp65 of cmv ( 5 b ). the identified stimulatory peptides fully matched with the information we already had from those donors with respect to their fine specificity profiles . our assay format on whole pbmc does not discriminate between cd4 and cd8 responses . therefore we demonstrated that by simple , one step removal of one of the two subsets with magnetic beads ( dynabeds , dynal , denmark ), we can achieve a clear - cut discrimination . three representative subjects are shown here . the left graphs show responses of unfractionated pbmc to a set of immunodominant cd4 or cd8 peptides for the various donors . as predicted , cd4 + cells ( middle panels ) only responded to cd4 peptides , but not to cd8 peptides , and viceversa ( right panels ). two children from whom small amounts of blood were available were screened for cellular immunity against a panel of relevant antigens . informative responses were obtained by cellelisa in 384 well plates , while conventional intracytoplasmic staining ( ics ) for ifng was occasionally negative and showed much lower stimulation indexes . as frequently occurs with pediatric samples , the number of pbmc was not sufficient to run an extensive screening by ics with the second patient ( 7 b ), while the miniaturized format allowed the complete screening with the panel of recall antigens . in the research setting , our assays provides information also on responsiveness and specificity of established t cell lines . the major advantage over conventional lymphoproliferation used for these studies is that no radioactivity is used , very few cells are needed and the result can be available in 24 hrs instead of 3 - 4 days . the figure shows experiments with t cell lines derived from naive precursors specific for hiv ( upper panels ) and t cell lines specific for recoll antigens ( pp65 of cmv ). wells were coated with a pool of 1st antibodies specific for il2 , il4 , il5 , ifng , tnfa , gm - csf . individual recombinant cytokines were titrated . a pool of 2nd biotinylated antibodies with the same specificities was added , followed by streptavidine - alkaline phosphatase and substrate . the individual titration curves show that the assay can reveal individual cytokines , without interference among the different antibody - cytokine systems . screening of polyelisa positive supernatants for individual cytokines supernatants from a cd4 t cell line ( pep30 ) and from a cd8 t cell line ( nv9 ) specific for immunodominant peptide of cmv were tested by polyelisa ( not shown ). the positive supernatants were tested for individual cytokines , as shown here . this confirms that polyelisa can detect either single cytokines ( fig9 ) or mixture of cytokines naturally produced by antigen - activated t cell . proteins : tetanus toxoid ( tt ) and ppd were purchased from statens seruminstitut , copenhagen , denmark ; cmv pp65 protein expressed in e . coli was provided by a . loregian , university of padua it ; cmv lysate and t . gondii lysate were purchased from abi , columbia , md . ; p . carinii suspension was provided by j - c . caillez , pasteur institute , lille , fr ; c . albicans , asp . fumigatus and cryptococcus were prepared in our lab as an autoclaved suspension . peptides of cmv pp65 and ie - 1 proteins , of sars coronavirus spike protein , of different hiv proteins and selected peptides from other cviruses were synthesized as 15 mers overlapping by 11 aa residues when used as comprehensive peptide panels . ab pairs from mabtech , stockholm se , were used at the indicated dilutions . the kits contain the first antibody for coating , the second biotinylated antibody and alkaline phosphatase conjugated streptavidin . standards were provided with the kits and prepared as instructed . pbmc preparation by standard ficoll gradient , resuspended at 10 6 / ml in rpmi 5 % fcs . human cd4 and cd8 t cell lines with different specificities were available in our laboratory . the lines have been produced as described in detail ( 7 , 8 ) plates were purchased from nunc , denmark . 96w plates were either flat bottom or round bottom for cultures , immuno - modules were used for elisa assays in 96 well format . 384 well and 1536 well plates were clear plastic tissue culture grade . they were used both for cell cultures and for elisa assays . for reading the elisa plates , we used the slt 340 atl , tecan , germany for 96w and the genios , tecan for 384w . since 1536w reader was not yet available , assays in 1536w plates were run as quadruplicates and four wells containing 10 μl pnpp were pooled after incubation in one well of a 384w plate . results are shown as od405 × 1000 at the indicated times of incubation at rt . for sample handling , eight channel electronic pipets ( eppendorf ) were used to transfer cells or elisa reagent in all plate formats . multiple six channel hamilton syringes with teflon coated plungers ( gas tight ) were used with a manual dispense when 1 μl samples ( antigens ) were distributed in 1536w plates . a multiwell automatic dispenser ( wellmate , matrix , hudson , n . h .) was used for processing of the 384w plates . pbmc prepared by conventional ficoll gradient were resuspended in rpmi1640 medium containing l - glutamin and 5 % fetal calf serum ( fcs ) or in 5 % human ab serum selected for low background in the elisa assays . preliminary tests , in fact , showed that sera from certain healthy subjects have detectable concentrations of several cytokines that interfere with the elisa assay by resulting in a high background . antigens were prepared as 10 × solutions and dispensed in wells before seeding pbmc . pbmc were brought to 10 6 / ml and dispensed at the indicated volumes by using multichannel pipets ( 200 μl for 96w and 50 μl for 384w plates ) or hamilton syringes ( 10 μl for 1536w plates ). plates were incubated overnight , supernatants were transferred to replica plates for later use and the culture plate was developed for cellelisa . the volumes of the reagents in the different plate formats are summarized below : reagent 96 384 1536 i ab , 1 μg / ml in pbs 100 20 5 saturation , pbs hsa 1 % 200 100 10 sample 200 50 10 ii ab , 1 μg / ml in pbs hsa 1 % 100 20 5 strep - alp , 1 : 200 in pbs hsa 1 % 100 20 5 pnpp 200 50 10 coating with i ab , on , rt , washing with pbs 4 × ( flushing with wash bottle ) saturation with hsa 1 % in pbs , 1 hrs , washing pbs 4 × ( flushing with wash bottle ) sample , 1 hr , washing with pbs 0 . 05 % tween 2 ×, pbs 4 × ( flushing with wash bottle ) ii ab , 2 hrs , washing with pbs 0 . 05 % tween 2 ×, pbs 4 × ( flushing with wash bottle ) strep - alp , 1 hr , washing pbs 0 . 05 % tween 2 ×, pbs 4 × ( flushing with wash bottle ) coating antibodies at 1 mg / ml specific for different cytokines ( il2 , il4 , il5 , ifng , tnfa , gm - csf , 20 μl each ) were pooled . for coating , 6 μl of the pool were added to 1 ml pbs . biotinylated antibodies specific for the same set of cytokines ( 20 μl each ) were pooled . when used as second reagent , 2 μl of this pool were added to 1 ml pbs 1 % hsa . alp - streptavidin was used at 1 : 2000 as indicated . culture wells ( 96 , 384 or 1536 well plates ) were handled under sterile conditions for coating with sterile i antibody . often on coating and 1 hr saturation with hsa 1 % in pbs , the liquid was removed by aspiration with a blunt needle connected to a vacuum source . antigens as 10 × solutions were dispensed in the plates ( 10 μl in 96 well plates , 5 μl in 384 , 1 μl in 1536 ) and let to dry for 1 hr under the laminar flow cabinet . the plates were coated with an adherent plastic sheet and stored at room temperature . pbmc were dispensed at the indicated volumes . after on incubation , supernatants were transferred to a replica plate and the wells were developed . t cell lines with cd4 or cd8 phenotype and specific for tetanus toxoid ( tt ), hiv env and several epitopes of cmv pp65 have been used as standards to run sensitivity experiments . by using established t cell lines ( 7 , 8 ), in fact , we know exactly the number of input antigen specific t cells . thus we can run limiting dilution experiments that are not feasible with pbmc , in which the actual number of specific cells can only be inferred by assays like tetramers , elispot or intracytoplasmic cytokine staining , assuming 100 % efficiency . 1 . kern f . li pira g , gratama j . manca f , roederer m . measuring antigen specific immune responses : understanding immunopathogenesis and improving diagnostics in infectious diseases , autoimmunity and cancer . trends immunol . 2005 , 9 , 477 - 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