Patent Application: US-34828694-A

Abstract:
this invention discloses a method for the treatment or prevention of autoimmune diseases , allergic or atopic disorders and graft rejection . specifically , it provides a means of killing a specific sub - population of t lymphocytes while leaving the majority of other t lymphocytes in the population unaffected . the sub - population of t lymphocytes are killed by repeatedly challenging the population with an antigen in conjunction with administration of interleukin - 4 .

Description:
the following detailed description of the invention is provided to aid those skilled in the art in practicing the same . even so , the following detailed description should not be construed to unduly limit the present invention , as modifications and variations in the embodiments herein discussed may be made by those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery . the disclosures of each of the references cited in the present application are herein incorporated by reference in their entirety . materials . female b10 . a and balb / c mice were purchased from charles river . pigeon cytochrome c , and propidium iodide , as purchased from sigma chemical co . ( st . louis , mo .). purified murine ril - 4 was kindly provided by dr . w . paul ( national institute of allergy and infectious diseases , nih ). the anti - murine il - 2 monoclonal antibody ( mab ) s4b6 . 1 was generously provided by dr . j . zuniga - pflucker ( national institute of allergy and infectious diseases , nih ). anti - murine cd3ε mab 145 - 2c11 ( 105 ) used in experiments was immobilized by coating either 12 - or 96 - well culture plates ( 500 μl or 100 μl , respectively ) ( costar , cambridge mass . ), at a concentration of either 1 μg / ml or 10 μg / ml in phosphate - buffered saline ( pbs ) for 120 minutes or overnight at 37 ° c . the plates were washed three times with medium ( click &# 39 ; s or eagle &# 39 ; s hank &# 39 ; s amino acid , ehaa , and 10 % fcs , 2 mm glutamine , 50 μm β - mercaptoethanol , penicillin and streptomycin ; biofluids , inc ., rockville , md .) before use . mabs specific for mhc class ii molecules ( a k , 10 . 2 . 16 ; and e k , y17 ), were a gift from dr . r . schwartz ( national institute of allergy and infectious diseases , nih ). cell culture . the murine nontransformed t cell clone a . e7 was carried as described previously ( 106 ). for experimentation , either resting cells (& gt ; 2 weeks following antigen stimulation ) or antigen stimulated cells were used . antigen stimulation consisted of culturing 1 × 10 6 resting t cells with 1 × 10 7 b10 . a irradiated ( 3000r ) splenocytes and 5 μm pigeon cytochrome c in 2 mls total volume . after 48 hours , the antigen presenting cells ( apcs ) were removed by mhc class ii mab mediated complement lysis ( low - tox - m rabbit complement , cedarlane laboratories , westbury , n . y . ), and the cells were recovered by lympholyte m density centrifugation ( cedar lane laboratories ) as previously described ( 41 , 104 ). the cells were then recultured for 48 hours in medium with 1 % mla ( gibbon ape leukemia cell supernatant containing 140 u / ml of il - 2 activity ) or ril - 4 ( 10 - 1000 u / ml ). t cells were then harvested and washed and assays were carried out in 96 - well flat - bottomed plates in triplicate , with 200 μl total volume for 48 hours . 5 × 10 4 cells / well ( with the exception of table i , experiment 1 which was 1 × 10 5 cells / well ) were added with the designated lymphokine , either in the absence or presence of t cell receptor stimuli . the live cell number was then determined as described below . t cell proliferation assays . a parallel culture of 5 × 10 4 cells , without t cell receptor ( tcr ) stimulation , was pulsed by the addition of 1 μci of 3 h ! thymidine ( 3 h - tdr ) ( 6 . 7 μci / mmol , new england nuclear ) for 16 - 24 hours . cells were subsequently harvested onto glass filter paper , and the samples counted by liquid scintillation on an lkb betaplate counter . data are expressed as the mean cpm of triplicates . cell viability . viable cell number was determined by manual counting of trypan blue excluding cells using a hemocytometer by flow cytometry ( facs ) with propidium iodide stained cells . for flow cytometry quantitation , cells were harvested by pipetting , washed once in phosphate buffered saline ( pbs ), and each sample was suspended in a constant volume of pbs with propidium iodide ( 2 μg / ml ). the fluorescence intensity of samples collected for a constant amount of time ( 100 sec .) was determined using a facscan ii analyzer with lysis ii software ( becton dickenson , mountain view , calif .). for this procedure , each sample is kept in a constant volume and the cells are collected for a constant amount of time , independent of the number of events . only the live cell number , as gated by forward scatter and propidium iodide exclusion , is quantitated . a comparison between duplicate cultures analyzed for live cell number by trypan blue exclusion or flow cytometric analysis reveals that the relationship between these two quantitation methods is linear , evidenced by an r value & gt ; 0 . 97 . 1 × 10 6 cells were incubated in 12 - well plates coated with anti - cd3ε mab for 48 hours , at which time the cells were harvested by gentle scraping and prepared by a modification of a previous procedure ( 8 ). briefly , cells were washed once in pbs and incubated in 20 μl of lysis buffer ( 50 mm tris , ph 8 . 0 , 10 mm edta , 500 μg / ml proteinase k , and 0 . 5 % sodium sarkosyl ) for 1 hour at 50 ° c . rnase a ( 50 μg / ml ) ( boehringer mannheim ) was added and the cells were incubated for an additional hour at 50 ° c . dye buffer ( 10 mm edta , 1 % ( w / v ) low melting point agarose , 0 . 25 % ( w / v ) bromphenol blue , and 40 % ( w / v ) sucrose ) was added , the samples were heated to 70 ° c . for five minutes , quenched on ice , and electrophoresed in a 2 % nusieve agarose , 1 % ultra - pure agarose gel with ethidium bromide . we have previously shown that il - 2 participates in an apparent feedback pathway , termed propriocidal regulation , by predisposing t lymphocytes to antigen - induced apoptosis ( 26 ). we therefore determined if il - 4 , another t cell growth factor , would induce this pathway . we first studied the nontransformed cd4 + t h 1 clone a . e7 that responds to pigeon cytochrome c in the context of an e k mhc class ii molecule . this clone has been shown to upregulate its il - 4 receptor in response to antigenic stimulation and proliferate in response to il - 4 ( 41 ). as shown in table 1 , antigen stimulated a . e7 cells proliferate to il - 4 in a dose dependent manner , as indicated by tritiated thymidine ( 3 h - tdr ) incorporation ( experiment 1 ). moreover , there was a dramatic cell loss when the proliferating cells were subsequently placed onto anti - cd3ε - coated plates for 48 hours , as compared to the uncoated plate control . the reduction in cell number was minimal with no growth lymphokine added and increased roughly in proportion to the degree of proliferation achieved with increasing amounts of lymphokine . cells treated with 1000 u / ml il - 4 showed an 84 % decrease in cell number following tcr stimulation . the overall cell loss found with il - 4 was as great as that obtained with il - 2 stimulation ( 85 % versus 87 %, respectively ). we observed a similar phenomenon with the lymphokine - dependent t cell lines , cr . 4r and ct . 4s . we could not detect any t cell receptor surface expression in either cell line and anti - cd3ε stimulation had no effect on these cells ( s . b . and m . l ., unpublished results ). nonetheless , when tcr occupancy was mimicked by a combination of phorbol myristic acetate ( pma ) and ionomycin , extensive cell loss was observed after 48 hours ( table 1 , experiment 2 ). greater than 90 % cell loss was observed for ct . 4r cells exposed to either il - 2 or il4 , and 87 % cell loss was seen for ct . 4s cells incubated with il - 4 . several features of the cell loss in these experiments suggested that cell death was occurring . first , microscopic examination in all cases revealed cells appearing to undergo apoptosis . as shown in fig1 trypan blue staining can detect non - viable cells ( dark colored ) in the cells stimulated with anti - cd3ε antibody more than in untreated samples . second , the number of a . e7 cells following 1000 u / ml il - 4 and anti - cd3ε stimulation was less than the number of cells put into the wells implying cell loss in addition to any potential block in proliferation ( table 1 , experiment 1 ). third , ladders of nucleosomal length dna were obtained following il - 4 and anti - cd3ε treatment of a . e7 cells , indicating the occurrence of apoptosis . as shown in fig2 dna fragmentation was observed in cells cultured with platebound anti - cd3ε mab ( lanes 3 and 4 ), and was not observed in cells cultured with medium alone ( lanes 1 and 2 ). we also observed cell death when il - 4 treated a . e7 cells were co - cultured with irradiated splenocytes and antigen ( table 1 , experiment 3 ). because il - 4 can stimulate the release of il - 2 under certain conditions ( 107 ), a mab capable of binding il - 2 ( s4b6 . 1 ) was included in the il - 4 stimulation . this did not inhibit subsequent t cell stimulation - induced apoptosis ( table 1 , experiment 4 ), suggesting il - 4 treatment alone predisposes t cells to apoptosis . because these experiments were carried out in t cell clones that had been carried in vitro for a long period of time , we investigated whether il - 4 could predispose lymph node cells to apoptosis . conditions for stimulating lymph node t ( lnt ) cells to produce lymphokines and proliferate in response to either il - 2 or il - 4 have recently been determined ( 108 ). treatment with tcr stimulation and il - 2 produces cultures exhibiting a predominantly tn1 phenotype producing and responding to il - 2 , whereas the inclusion of il - 4 leads to a t h 2 phenotype of cells producing and responding to il - 4 ( 108 ). freshly isolated lymph node cells were treated for 72 hours with either soluble anti - cd3ε mab or concavalin a , and il - 2 or il - 4 . the lnt cells proliferated significantly in response to lymphokine in all samples ( table 2 , cpm ). there was large decrease in the number of live cells recovered following a 48 - hour incubation on anti - cd3ε - coated plates compared to the plastic control at all conditions tested ( table 2 ). these results show that il - 4 has the ability to predispose lnt cells to apoptosis . furthermore , as was previously observed with il - 2 ( 26 ), il - 4 by itself can evoke the propriocidal pathway that leads to apoptosis following antigen receptor stimulation . table 1__________________________________________________________________________the effect of il - 4 and t cell receptorstimulation on t cell viability . exp . cells pretreatment . sup . 1 cpm control % cell loss__________________________________________________________________________ cell number (× 10 . sup .- 5 / ml ) anti - cd31 a . e7 none 2 , 060 7 . 3 ± 0 . 7 5 . 4 ± 0 . 7 27 a . e7 14 u ml . sup .- 1 il - 2 173 , 845 37 . 3 ± 4 . 1 4 . 7 ± 0 . 4 87 a . e7 10 u ml . sup .- 1 il - 4 2 , 275 6 . 3 ± 0 . 3 4 . 7 ± 0 . 4 25 a . e7 100 u ml . sup .- 1 il - 4 17 , 833 10 . 1 ± 0 . 8 5 . 5 ± 0 . 8 45 a . e7 1000 u ml . sup .- 1 il - 4 89 , 159 18 . 6 ± 0 . 4 3 . 0 ± 0 . 4 84 pma / i . sup . 22 ct . 4r 28 u ml . sup .- 1 il - 2 313 , 574 55 . 3 ± 5 . 4 2 . 9 ± 0 . 9 95 ct . 4r 1000 u ml . sup .- 1 il - 4 317 , 227 34 . 3 ± 4 . 6 3 . 6 ± 0 . 8 90 ct . 4s 1000 u ml . sup .- 1 il - 4 155 , 982 27 . 7 ± 1 . 8 3 . 6 ± 0 . 7 87 facs cell number . sup . 3 - ag + ag3 a . e7 1000 u ml . sup .- 1 il - 4 257 , 568 22 , 703 5 , 348 76 anti - cd3e4 a . e7 14 u ml . sup .- 1 il - 2 266 , 137 11 . 7 ± 1 . 6 2 . 6 ± 0 . 4 78 a . e7 1000 u ml . sup .- 1 il - 4 257 , 913 9 . 4 ± 1 . 7 1 . 9 ± 0 . 3 80 a . e7 1000 u ml . sup .- 1 il - 4 + s4b6 . 1 257 , 568 9 . 3 ± 1 . 3 2 . 0 ± 0 . 5 79__________________________________________________________________________ . sup . 1 indicates the treatment of a . e7 cells following 48 hour antigen stimulation . ct . 4r and ct . 4s cell lines did not undergo antigen stimulation but were pretreated as described . the concentration of lymphokine indicated was kept constant for each sample during the 48 hour pretreatment and the 48 hour duration of the experiment . . sup . 2 these cells do not express tcr on the cell surface , so were cultured with pma ( 10 ng / ml ) and ionophore ( 2 μm ). . sup . 3 indicates the live a . e7 cell number as determined by forward scatter profile , propidium iodide dye exclusion and surface staining with antimouse vαl 1 mab ( pharmingen ) ( see materials and methods ). experimental conditions consisted of a 10fold excess of b10 . a irradiated spleen cells , 1 μm pigeon cytochrome c (+ ag ) and 30 μg of antimouse il2 μab s4b6 . 1 . table 2______________________________________the effect of lymphokines and antigen receptorstimulation on lymph node cell viability . pretreatmenttcr lymph - cell number cellstim . okine cpm control anti - cd3e loss______________________________________anti - il - 2 85 , 912 16 , 876 ± 3 , 775 7 , 917 ± 1 , 310 53cd3εcon a il - 2 54 , 835 28 , 226 ± 660 8 , 489 ± 2 , 804 70anti - il - 4 93 , 725 42 , 920 ± 3 , 409 4 , 628 ± 475 89cd3εcon a il - 4 81 , 562 36 , 930 ± 2 , 705 3 , 510 ± 887 91______________________________________ balb / c lymph node cells were cultured at a concentration of 1 × 10 6 cells / ml for 72 hours with soluble anti - cd3ε ( 3 μg / ml ) or concavalin a ( 3 μg / ml ) in the presence of il - 2 ( 14 u / ml ) or il - 4 ( 1000 u / ml ). the cells ( 5 × 10 4 cells / well ) were washed extensively and incubated with medium or anti - cd3 - coated plates for an additional 48 hours in the presence of lymphokine . the cells were then harvested and the live cell number was determined by facs analysis . we previously proposed the term propriocidal regulation for the antigen receptor - stimulated apoptosis of mature t lymphocytes that were induced into the cell cycle by il - 2 ( 26 ). this mechanism would result in the elimination of any t lymphocyte that had a sufficient affinity for the inciting antigen . our previous results suggest that in order for the t lymphocyte to undergo propriocidal cell death , it must be responding to il - 2 treatment , but not necessarily producing the lymphokine . we now extend these results to show that a . e7 , a t h 1 clone that can respond to , but not produce il - 4 , will undergo apoptosis by tcr stimulation if actively cycling in response to il - 4 treatment . we have also shown that normal lymph node cells driven into cell cycle by antigen receptor stimulation and either il - 2 or il - 4 treatment , undergo cell death upon subsequent tcr stimulation . cell cycling per se is not required , because we have found that certain blocking agents do not prevent tcr - mediated apoptosis ( 27 ). nonetheless , agents that prevent progression beyond late g1 , and not those that block proliferation in s phase , were found to be capable of inhibiting apoptosis ( 27 ). thus , we favor the hypothesis that cell cycle progression beyond late g1 , stimulated by growth lymphokines such as il - 2 or il - 4 , is permissive for tcr - mediated death in t lymphocytes . it is likely that cell cycle progression beyond the late g1 stage due to the transformed phenotype of t cell hybridomas and lymphomas accounts for their sensitivity to tcr - mediated apoptosis without lymphokine treatment ( 13 - 18 ). our results suggest that an intrinsic property of the t lymphocyte response to a growth lymphokine such as il - 4 is the susceptibility to apoptosis upon further tcr stimulation . moreover , this response portrays a mechanism by which an immune response to specific antigens may be naturally suppressed . as shown in fig3 immunization with a specific peptide or protein is carried out on day one . in the case of multiple sclerosis , for example , there is evidence that either of two immunodominant peptides from myelin basic protein ( mbp ) are encephalitogenic in man ; mbp 84 - 102 ( the preferred peptide ), or mbp 143 - 168 ( 78 , 79 ). either or both of these peptides , coupled to tetanus toxoid , can be given in alum adjuvant intramuscularly ( im ), at a dose between about 10 to about 1000 μg . early immunization experience using proteins or peptides has suggested that intramuscular administration is optimal ( 109 - 113 ). newer data suggest that oral administration may also be effective ( 94 ). as with any medicinal substance , or biologic , tests on any peptides and proteins used for the immunization would need to be routinely carried out over a range of doses to determine : 1 ) the pharmacokinetic behavior of these substances ; 2 ) their immunogenicity ; and 3 ) safety and identification of any untoward effects . this would constitute a phase i clinical trial ( 114 ). thus , the particular proteins or peptides employed in this protocol ( for example , in multiple sclerosis , mbp 84 - 102 , or mbp 143 - 168 ; in uveitis , the s antigen ; or in rheumatoid arthritis , type ii collagen ) would require individual routine optimization . similar intervention could be used with preparations of allergy - inducing proteins . these could be derived from a variety of allergen protein extracts that are now used clinically , or could be generated by recombinant dna technology for those such as hornet venom antigen 5 , for which cloned dna is available ( 100 ). ample evidence from the development of vaccines suggests that either synthetic peptides or recombinant dna - derived proteins are effective in eliciting an immune response in humans ( 109 - 112 ). these studies also provide guidance as to the range of doses effective for immunization . 1 ) hepatitis b surface antigen , produced as a recombinant protein in yeast . adults 2 . 5 to 20 μg ; children 1 . 25 to 5 μg intramuscularly ( im ). 90 - 96 % of vaccines showed an immune response , with the best response at 10 - 20 μg ( 109 ). further studies showed the efficacy of a 10 μg dose , with better results when given im rather than subcutaneously ( 110 ). 20 μg doses in alum adjuvant given im were found to be effective at preventing infection in clinical trials ( 111 ). 2 ) hiv gp 120 , either natural or recombinant molecules . doses in chimpanzees between 50 - 1000 μg elicit t cell responses ( 115 ). 1 ) chorionic gonadotropin . several studies have indicated successful immune responses against a human chorionic gonadotropin - β subunit peptide ( residues 109 - 145 ) coupled to cholera or tetanus toxoid and given in doses from 50 - 1000 μg in alum adjuvant ( 112 ). 2 ) malaria sporozoite antigen . studies of a plasmodium falciparum peptide ( nanp ) 3 coupled to tetanus toxoid showed an immune response to doses of 20 - 160 μg of peptide conjugate given im , with the best response at 160 μg ( 113 ). immunization is then followed by a waiting period during which the antigen activates the subset of t cells bearing reactive tcrs , causing them to express il - 4 receptors and possibly il - 4 . this process will only upregulate il - 4 receptors on cells that have been antigenically - stimulated ( 36 ). based on studies of both human and mouse t cells in vitro , between about 12 to about 24 hours after antigen exposure are required to express significant increases in the numbers of il - 4 receptors , and as long as about 72 hours are required to express optimal numbers of lymphokine receptors on the majority of t cells ( 36 ). thus , the waiting period can be as short as about 12 hours or as long as about 72 hours , becoming increasingly optimal toward the upper end of this range . this is then followed by an infusion of high doses of il - 4 . though only very limited data exists on the clinical use of il - 4 ( 89 - 92 ), a great deal of information has been obtained from clinical studies using il - 2 . the administration of high - doses of the related t cell growth lymphokine il - 2 to humans has been well - studied in cancer patients , and various doses have been evaluated ( 116 - 120 ). data indicate that il - 2 should be given intravenously ( i . v .) either as frequent bolus doses or as a continuous infusion ( 116 - 118 ). doses that have been previously established range between about 300 to about 3000 units / kg / hour continuous infusion , or from 10 4 to 10 6 units / kg i . v . bolus ( 117 ). units are defined by standards available from the biological response modifiers program at the national institutes of health , and are defined as the quantity of il - 2 or il - 4 that gave 50 % maximal thymidine incorporation in the bioassay under standard conditions . side effects of these doses included chills , fever , malaise , headache , nausea and vomiting , weight gain due to fluid retention , diarrhea , rash , and pruritis , which can all be treated with acetaminophen or indomethacin ; no serious morbidity or mortality was observed . studies of il - 4 administration to humans used human recombinant il - 4 of specific activity 1 . 5 × 10 7 units / ug , given in doses of 10 - 20 ug / kg body weight , three times / day . ( 89 - 91 , 120 , 121 ) the side effects with il - 4 were similar to those observed with il - 2 and included weight gain due to water retention and nausea . after il - 4 treatment , the patient can be immediately reimmunized with an equivalent dose of antigen . for example , for multiple sclerosis , treatment can be carried out with about 10 to about 1000 μg of peptide mbp 84 - 102 coupled to tetanus toxoid and given in alum adjuvant im . it is likely that the preferred dose would be near the upper end of this range since greater tcr stimulation produces a greater level of apoptosis ( 26 , 27 ). il - 4 treatment would have stimulated the t cells bearing il - 4 receptors -- predominantly the disease - causing t cells -- and these cells would then be re - stimulated through their tcr . these cells will then undergo apoptosis . after an immunization period of about 12 to about 72 hours , the cycle would begin again with reinfusion of il - 4 . as will be described below , increased efficacy would likely result from multiple cycles of therapy . the treatment endpoints would be : i ) elimination of in vitro reactivity to the antigen , which can be easily measured where possible by various mixed lymphocyte or proliferation assays using peripheral blood lymphocytes ; ii ) amelioration of clinical symptoms ; or iii ) toxicity . the treatment endpoints for allergic diseases would be : i ) improvement of clinical symptoms ; ii ) normalization of an allergic skin test ; iii ) reduction in serum ige levels ; and iv ) where possible to measure , reduced t cell responses to the allergenic protein . several features of the present therapy require further explanation . first , it is expected that t cells besides those antigenically stimulated may express high affinity il - 4 receptors . however , this should not diminish the specificity of the therapy because only those cells whose tcrs are stimulated by rechallenge with antigen will undergo apoptosis , as described supra . the effectiveness of the therapy could be variable depending on the nature of the antigen and the exact protocol employed . extensive in vitro studies indicate that between 50 - 80 % of the antigen - specific il - 4 stimulated t cells will undergo apoptosis when rechallenged by tcr stimulation ( supra , 27 ). second , the reduction in number of antigen - specific t cells determines the overall effectiveness of the therapy . therefore , repeated cycles can substantially increase efficacy even if the level of killing in each cycle is only 50 - 70 % ( table 3 ). as shown in the mouse studies , supra , the level of antigen - reactive t cells will decrease below the number of such cells prior to the first immunization with repetitive immunization . furthermore , the expected toxicity of this protocol at moderate doses or lymphokine should be minor , and previous studies of the therapeutic use of growth lymphokines such as il - 2 or il - 4 in humans indicates that all side effects dissipate promptly following discontinuation of lymphokine treatment ( 89 - 91 , 116 , 117 ). the most serious side effect , fluid retention , should be minimized by the intermittent nature of il - 4 treatment . the 2 - 3 day rest period between doses would allow for diuresis of the fluid built up during il - 4 administration . finally , the repeated administration of antigen will cause production of some endogenous il - 4 , which will predispose some cells to apoptosis . while it is extremely unlikely that endogenous levels can reach the very high levels of il - 4 that can be administered pharmacologically , it is possible that empirically - determined decreases in the il - 4 dose could be achieved because of endogenous il - 4 effects . the level of killing is dependent on the total level of il - 4 to which the t cell is exposed , and this will reflect a combination of endogenous and exogenous sources ( supra , 26 , 27 ). with certain antigens , the pre - disposition of cells to apoptosis may be sufficiently induced by the endogenous production of il - 4 . in these cases , appropriate immunization with antigen , in the absence of exogenously administered il - 4 , could produce t cell apoptosis and a protective effect . based on the studies of the timing of susceptibility to apoptosis disclosed supra , immunizations repeated at specific intervals would be crucial for effective therapy . to effect il - 4 - mediated apoptosis , immunizations would have to be repeated at about 24 to about 120 hour intervals , preferably at about 24 to about 72 hour intervals , and would have to be repeated multiple times using antigen doses at about the high end of the ranges discussed above . t cell reactivity or cell - mediated immunity for the specific antigen could then be monitored by in vitro assays to determine that t cells had undergone apoptosis . absent the knowledge provided by the discovery disclosed herein , previous attempts to decrease immune responsiveness by repetitive immunization have not been optimal . for example , donor transfusion protocols to ameliorate graft rejection involved 3 transfusions given at 2 week intervals ( 122 , 123 ). allergy shots , i . e ., desensitization therapy , are typically given initially at 4 - 7 day intervals , after which intervals are progressively increased in length to 2 to 4 weeks ( 97 ). based on the present novel understanding of t cell apoptosis , the most effective immunization protocol would involve repetitive administrations of antigen at about 24 to 72 hour intervals . table 3______________________________________theoretical number of reactive cells afterfractional killing using il - 4 and t cellreceptor stimulation fractional reactive cellscycle killing remaining______________________________________start none 100 , 0001 70 % 30 , 0002 70 % 9 , 0003 70 % 2 , 7004 70 % 8105 70 % 2436 70 % 73______________________________________ theoretical values are based on starting with 100 , 000 cells and a constan killing efficiency of 70 %. a reduction of over 100fold is seen in 4 cycle and over 1000fold in 6 cycles . at a fractional killing of 50 %, a reductio of nearly 100fold would be seen in 6 cycles . a first order kinetics is represented here because the process of apoptosis involves a single letha hit deiivery as has been shown for apoptosis induced by antimetabolites ( 1 - 5 ). in medical procedures in which tissue is transferred between individuals who are genetically non - identical at their relevant histocompatibility antigen loci , herein referred to as allografting , and the transplanted tissue referred to as an allograft , the major problem encountered is rejection of the donor allograft by the host . the term &# 34 ; host &# 34 ; refers to the individual who is the recipient of the allograft , and the term &# 34 ; donor &# 34 ; refers to the individual from whom the allograft is derived . studies of the process of graft rejection have shown that it is due to the antigen - specific activation of t lymphocytes , especially those bearing cd8 surface molecules ( 124 ). more importantly , agents that block the ability of t cells to mount an immune response in humans effectively prevent or lessen graft rejection ( 125 ). since cd8 + t cells have been shown to be susceptible to apoptosis by il - 4 , supra , this phenomenon can be used as a specific means to eliminate the reactive t cells , thereby avoiding graft rejection . essentially the same protocol with respect to timing and il - 4 dose can be used for this therapy as was described supra for the therapy of autoimmune diseases . the major difference between this therapy and that described above is the source of antigen . major histocompatibility complex ( mhc ) antigens are cell surface proteins that are tremendously polymorphic among individuals . each individual &# 39 ; s cells bear a genetically determined set , or haplotype , of such antigens which serve as an immunological &# 34 ; fingerprint &# 34 ; on each cell ( 126 ). this allows one &# 39 ; s immune system , in particular those responses generated by t cells , to recognize one &# 39 ; s own cells , and to attack only cells that do not bear the self &# 34 ; fingerprint &# 34 ; ( 127 ). there are two classes of mhc -- class i antigens , found on all cells in the body ; and class ii antigens , found predominantly on monocytes , macrophages , b lymphocytes , dendritic cells , and activated t cells ( 126 ). it is the class i mhc antigens that are recognized by cd8 + t cells that are the predominant influence in allograft rejection ( 124 , 127 ). because of this complexity of mhc antigens , the simplest source is cells from the allograft donor . it has been empirically observed that transfusion of a graft recipient with donor blood suppresses graft rejection , although the mechanism of this effect is unknown , and the clinical effectiveness in many cases is modest ( 123 ). these protocols provide evidence that three transfusions of 200 ml of whole blood or packed cell equivalent from the donor is easily tolerated by the recipient with minimal side effects ( 122 ). there is evidence that the donor - transfusion in some cases elicited sensitizing antibody responses in the allograft host , and these patients were not given allografts ( 122 ). these studies possibly represent an empirical observation that pre - exposure to donor antigen suppresses the t cell response , although this is controversial ( 124 ). the present method includes administration of blood as a source of mhc antigens in doses of about 50 to about 200 ml to patients in cycle with il - 4 , as indicated in fig4 . in the case of kidney transplants , the amount of blood could be determined by the fluid tolerance of end - stage renal disease patients . the blood can be given as either whole blood , packed cells , or washed packed cell transfusions ( 123 ). the success of treatment can be assessed by : i ) a decreased requirement for general immunosuppressive medications ; ii ) graft survival ; and iii ) adequate function of the allograft . for example , the function of a transplanted kidney can be established by determining serum levels of creatinine and blood urea nitrogen ( 125 ). this can be followed by il - 4 infusion and rechallenge with blood cells as antigen as shown in fig3 . the invention being thus described , it will be obvious that the same may be varied in many ways . such variations are not to be regarded as a departure from the spirit and scope of the invention , and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims . 1 . kerr , j . f . r , and b . v . harmon . 1991 . definition and incidence of apoptosis : an historical perspective . in apoptosis : the molecular basis of cell death , l . d . tomei and f . o . cope , ed . cold spring harbor laboratory press , plainview , n . y ., p . 5 . 2 . lockshin , r . a ., and z . zakeri . 1991 . programmed cell death and apoptosis . in apoptosis : the molecular basis of cell death . l . d . tomei and f . o . cope , ed ., cold spring harbor press , plainview , n . y ., p . 47 . 3 . cohen , j . j ., r . c . duke , v . a . fadok , and k . s . sellins . 1992 . apoptosis and programmed cell death in immunity . ann . rev . immunol . 10 : 267 . 4 . duvall , e . and a . h . wyllie . 1986 . death and the cell . immunology today 7 : 115 . 5 . cotter , t . g ., s . v . lennon , j . g . glynn , and s . j . martin . 1990 . cell death via apoptosis and its relationship to growth . development and differentiation of both tumor and normal cells . anticancer research 10 : 1153 . 6 . von boehmer , h . 1988 . the developmental biology of t lymphocytes . ann . rev . immunol . 6 : 309 . 7 . marrack , p ., and j . kappler , 1987 . the t cell receptor . science 238 : 1073 . 8 . smith , c . a ., g . t . williams , r . kingston , e . j . jenkinson , and j . j . t . owen . 1989 . nature 337 : 181 . 9 . shi , y ., r . p . bissonnette , n . parfrey , m . szalay , r . t . kubo , and d . r . green . 1991 . in vivo administration of monoclonal antibodies to the cd3 t cell receptor complex induces cell death ( apoptosis ) in immature thymocytes . j . immunol . 146 : 3340 . 10 . mcconkey , d . j ., p . hartzell , j . f . amador - 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