Goulmy, E.A.J.M.

Horai, S.

Rood, J.J. van

Leiden University Scholary Publications

Cytotoxic Τ Lymphocytes Directed AgainstHLA-Bw35-linked Target Determinants ShowDifferences in Sensitivity toward Antibioticsduring Sensitization PeriodSatoshi Horai, Eis Goulmy, and Jon J. van RoodABSTRACT Recently u e demonstrated that the outcome of human CML* was mfluenced by the presenceof anttbiotics in the culture medium In earher itudies we hadfound that cytotoxic 7 lymphocytescould recognize HLA-Bw:i5-linked target determinants These so called Bw35 α and b deter-minants showed Mendehan segregationWe descnbe in this article α difference between the Bw35 α and b cytotoxic Τ cells in thcirsensitivity touatd antibiotus The lysii against the Βιν^5 b cytotoxic determmant ivas notmfluenced by either the presence or the absence of antibiotics during the sensitization periodwhereas the lysis against the Bw35 α cytotoxic determmant was drastically dimimshed whenthe tffector cells wire cultured in the absence of antibiotics during the sensitization periodABBREVIATIONSCML cell mediated lympholysis MLC mixcd lymphocyte cultureCTL cytotoxic Τ lymphocyte PHA phytohemagglutinmI N T R O D U C T I O NAntibiotics such as penicillm and streptomycin are widely used in tissue culturemedia to prevent bactenal infection This IS also the case in human cell typingtechniques such as MLC* and CML* Recently, we demonstrated that the outcomeof a human CML test was mfluenced by the presence of antibiotics in the culturemedium Allogenic CTLs* cultured in the presence of antibiotics during theinduction phase showed significantly stronger lysis against the specific target cellsin companson to CTLs cultured in the absence of antibiotics Furthermore, insome cases the CML assay was positive (according to the European CML Work-shop cntena) [1,2] only when antibiotics were added to the cultures, whereasno lysis could be detected in the absence of antibiotics [3]In 1976, we descnbed the reaction pattern of two CTLs that recognized adetermmant linked to but different from HLA-Bw35 and Bw53 These effectorcells were raised in vitro between A-, B-, and C-identical but D-nonidenticalsibhngs [4]Trom tbe Department of Immunohaematology Unnersity Medical Centre Leiden The NetheilandsAddress requests for rcpnnts to Eh Goulmy Department of Immi/nobaemato/og) Unitersitt MedicalCentte Rijnsburgerueg 10 23 53 AA Leiden The NetherlandsRecaied 1981Hurrun Immunology 4 335-341(1982)© Elsevicr Science Publishing C ο Ine 1982 3^552 Vanderbilt Ave New York NY 10017 0198 8859/82/0 10335 0^S2 ""5336 S Horai et alThe demonstration that antibiotics mfluence the outcome of CML has promptedus to investigate the effect of antibiotics on the generation of CTLs directedagamst HLA-Bw35/53-linked target determinantsMATERIALS AND METHODSLymphocyte donors Healthy HLA-typed panel donors were used Blood samplesfrom family ν R (2 parents and 13 children) were obtained at three differentbleeding times The HLA genotypes of this family are shown in Figure 1Lymphocyte preparation Penpheral blood lymphocytes were obtained from hep-armized blood separated by Ficoll—Isopaque gradient centnfugation The Sepa-ration and cryopreservation procedures were performed in antibiotic-free mediaof intrafamihal CTLs Responder cells (107) and 107 stimulator cells(irradiated with 2000R) were cultured in 25-cm2 tissue culture flasks in RPMI1640 medium contaming 15% pooled AB serum supplemented with 25 mMHEPES and 3 mM L-glutamine When antibiotics were used, the Standard amountsof penicillin (100 IU/ml) and streptomycin (100 μg/ml) were added to the cul-tures The responder and stimulator cells were cultured for 6 days at 37°C in ahumidified CO 2 incubator Thereafter, effector cells were collected and the CMLassay was carned outTarget celU Target cells were cultured for 6 days with or without antibioticsAfter 3 days 50 μΐ of PHA*-M stock solution (Difco) was added On day 6 thetarget cells were labeled with 100 μ θ of 5 I Cr for 1 hr at 37°C and washed threetimes before useFIGURE 1 The HLA genotyping of family vR (J) HLA-Α HLA-C crossing-over, theHLA type of this child IS A2, Bw35, Bw51, Cw2, Cw4, DR2, LB5 (2) LB5 and LB5x8are Leiden local assignments of DR5 sphtscdac bod ac bc ad bd bd bc ad ad bd ad ac2 )haplotypesa Α 2 Cw4 Bw35 LB5x8b Α 2 Cw4 Bw35 LB 5 2 )c Α 2 Cw5 Bw44 DRw8d A11 Cw2 Bw51 DR 2Influence of Antibiotics on Human CML 337CML assay CTLs and target cells were suspended in antibionc-free culture me-dmm Viabihty was assessed, and both CTL and target cell suspensions wereresuspended at the desired concentranons The CTLs and target cells were ιη-cubated together in round-bottom microtiter plates for 4 hr at 37°C After incubation the supernatants were collected for gamma counting In most instances,tnphcate wells were tested in at least four effector cell dilunons When cellnumbers were hmited, only one effector/target ratio (60 1 or 50 1) was usedSpontaneous release was determined from the wells with target cells incubatedin culture medium alone Maximum release was obtained from Zaponin (CoukerElectronics Ltd , Herts, England)-RPMI 1640 lysis of the target cells The per-centage of lysis was calculated according to the following formulaiiiagc ui lyais was Ltueuidicu aetoruing to uie ronowiiig lumiiuamean of expenmental release — mean of spontaneous releasemean of maximum release — mean of spontaneous releaseχ 100Percentages equal to or below 10% lysis were considered negative when onlyone effector/target cell ratio was used Standard errors of the mean of tnphcateswere always less than 5%RESULTSFamily StudiesThe HLA genotyping of family ν R IS shown in Figure 1 Both paternal haplotypescarry the same serologically defmed HLA-A, B, and C anngens but differ for theDR antigens The father is heterozygous for D as shown by MLC studies [4]Consequently, two paternal haplotypes can be identined, and CTLs can be ιη-duced between sibhngs shanng the same maternal haplotype (see Fig 1) Recip-rocal lysis was obtained between the sibhngs that differed only in the paternalhaplotype An example of reciprocal lysis and Mendehan segregation is shownin Table 1 Furthermore, the influence of the presence of antibiotics in the culturemedium on the outcome of specific target cell lysis is presented as well ReciprocalCTL combinations were induced between siblings C3 and C8, cultured eitherwith or without antibiotics dunng the sensitization phase As shown in Table 1,CTLs C3 C8x cultured in the presence of antibiotics showed lysis (varying be-tween 39% and 53%) against target cells that carried the b haplotype WhenCTLs C3 C8x were generated in the absence of antibiotics, the lysis was equallystrong In contrast, the reciprocal combination C8 C3x, sensitized in the presenceof antibiotics, showed lysis against target cells that carried the a haplotype varyingfrom 27% to 33%, whereas the same CTLs sensitized in the absence of antibioticsgave only 12-20% lysis No positive CML was obtained by both CTLs againstautologous target cells or target cells that did not carry the corresponding hap-lotypesResults similar to those shown in Table 1 were obtained in the second ex-penment using different bleedings from the family members (data not shown)Although the CTLs showed a lower degree of lysis in the second expenment, ltwas clear that the presence of antibiotics did not affect the generation ofCTLs C3 C8x, whereas in the case of CTLs C8 C3x all combinations showed aieduction in lytic acnvity in the absence of antibioticsTo determine whether specific lysis obtained with CTLs C8 C3x was signtfi-cantly different when antibiotics were absent or present, additional expenmentswere set up and effector cell titrations carried out (Fig 2) CTLs were inducedin either the presence or the absence of antibiotics and tested against target cells338TABLE 1 CML results in family ν RTarget cellhaplotypesSpecific 51Cr releasc'C3 C8x C8C3xC3 (ac)C8 (bc)F(ab)Μ (cd)Cl (ac)C2 (bc/d)C4 (bc)C5 (ad)C6 (bd)C7 (bd)C9 (a643533248448463948536243561054534338334279103210930185ΙόNT''12NTNT20NTNTTThc pc'C TLs w.CIXs'Not tested>crccnt lysis o( CTLs C? C8x and C8 O x are givcn for rcspecnvely 50 1 and 60 1 cffcctor/targct ratiostre induccd in the presence of annbujtics and testtd against carget ctlls eultured wirh antibioticswere mduteci in the absenet of mtibiotics and tested against target cells eultured without antibioticsFIGURE 2. Reacuon patterns of two mtrafamihal CTLs against specific target cells ineither the presence or the absence of antibiotics CTLs were induced in the presence ofantibiotics and tested against specific target cells eultured with antibiotics (B) and withoutantibiotics ( # ) CTLs were induced in the absence of antibiotics and tested against specifictarget cells eultured with antibiotics (D) and without antibiotics (O) (a) CTLs C3 C8x,(b) CTLs C8 O x50-40Hra0),_ 30Οο.(Λ2 0 -1050-4 0 -3 0 -2 0 -1 0 -6 1 12 1 25 1 50 1 7 1 15 1 30 1 60 1E f f e c t o r t a r g e t r a t l o sInfluence of Antibiotics on Human CML 339cultured with or without antibiotics In all titrations, the absence of antibioticsdunng generation of the CTLs C8 C3x (anti-a-haplotype) diminished the specificlysis (Fig 2b), whereas the absence of antibiotics had no influence on the gen-eration of CTLs C3 C8x (anti-b haplotype) (Fig 2a) Furthermore, an influenceof antibiotics on target cell cultures could not be demonstratedPanel StudyNext the difference in susceptibihty to antibiotics between the two HLA-Bw35CTLs was tested against an unrelated panel selected for the presence or absenceof the Bw35 antigen Reciprocal CTLs were raised between two other familymembers, namely, child 1 and child 4 As shown in Figure 1, child 1 and child4 differ only in the paternal (Α-, Β-, and C-identical) haplotype Our earherobservation [4,5] of subdividing the serological defined Bw35 by CTLs led usto divide Bw35-posmve panel members mto two groups, called Bw35 a and bTable 2 shows the percentage of lysis by the CTLs against 7 family members and19 unrelated panel members When the CTLs that specifically lyse the Bw35 awere cultured in the presence of antibiotics, target cells from family members(Cl, F, C3, and C5) and unrelated panel members (1—7) were lyzed However,when no antibiotics were present in the sensmzation phase, in each case lysiswas diminished In contrast, no influence of antibiotics could be detected on thelevel of lysis when anti-Bw35 b-type CTLs were tested against Bw35 b-positivetarget cells (C4, F, C6, C8 and panel members 8-15) No lysis was obtained onthe Bw35-negative target cells (panel members 16-19) m either the presence orthe absence of antibioticsDISCUSSIONIn serology, the effects of chemotherapeutics and antibiotics on HLA typing havebeen descnbed [6—11] Recently, a report from our laboratory demonstratedthat, when lymphocytes were incubated with pemcillin and washed before HLAtyping, the cytotoxic reactivities of some typing sera with their correspondingantigen were blocked [12]Furthermore, one of us showed that antibiotics such as pemcillin and strep-tomycin could influence the specificity of CTLs in some responder—stimulatorcombinations [3] This IS also the case in one type of HLA-Bw35-specific CTLs,as demonstrated in the present studyThe two CTLs that showed speaficity for HLA-Bw35-hnked target determi-nants were induced in vitro between A-, B-, and C-identical siblings In this studywe were able to demonstrate the difference in antibiotic susceptibility betweenthe two different Bw35 CTLs Although we observed in different expenmentsvariable degrees of specific lysis, in all expenments lysis by anti-Bw35 a CTLswas diminished when CTLs were induced in the absence of antibiotics Thepresence of a Standard amount of pemcillin and streptomycin dunng the 6-daysensitizanon phase seemed to be most cntical This difference was not due tothe infe-tion of MLC cultures without antibiotics, since no bactena were foundin these cultures The presence of antibiotics in the target cell cultures did notinfluence specific lysis Once CTLs were induced, a neghgible difference in spe-cific 1 ysis intensity was obtained whether target cells were cultured with or withoutantibiotics (Fig 2) We observed no influence of antibiotics on [ iH]thymidineincorporation in MLC cultures [3]The mechanism by which the presence of antibiotics duTing the inductionphase induces stronger cytotoxic effector cells than in the absence of antibiotics340 S. Horai et al.TABLE 2 CML results against family and unrelated panel membersTarget cells"FamilymembersClC4FC3C5C6C8Unrelated panel12345678910111213141516171819ClC4x+- 62640103026- 172531745353746323030300102_ä-23143η44134081421750334243393534322111Speofic 5 lCr release4C4.Clx+220363229- 30232423121518295 .02401023564-6- 6211316Ί- 3131513781117400001114353Bw35 type'aba + baabbaaaaaaabbbbbbbb_/--''Ali targct cclls wcre cultured in thc presence of antibiocics'The percentages lysis of CTLs Cl C4x and CM Clx aregiven for, respectively, 50 1 and 60 1 effector/urget latiosCTLs were induccd in thc presence of antibiotics''CTLs were induced in the absence oi antibioncs'Thc HLA-Bw35 a and b types were estabhshed in earher studies [4,5]'HLA-Bw?5-negative individualsis at the moment still unclear. Several possibilities explaining these observationscan be considered.One possible explanation is that the combination of drugs and some HLAdeterminants during the induction phase of CTLs is more immunogenic thanHLA determinants alone. Α second possibility is that the drug renders the re-sponder cells more efficient in recognizing some antigenic determinants duringthe induction phase of CTLs.Whatever the mechanism is, it is clear that the presence of antibiotics in theinduction phase can drastically alter the CML specificities. According to theEuropean CML Workshop criteria, percentages of lysis equal to or below 10%are considered negative {1,2}. In the present study, we observed in all cases aInfluence of Antibiotics on Human CML 341reduction in lysis in antibiotic-free cultures of HLA-Bw35 a-type CTLs, amongthese, some of the combinations even became negative (Table 2) This raises avalid argument for investigating the overall influence of antibiotics in cytotoxicassays Further studies are needed to investigate the drug effect on the sensiti-zation phase of CTLs directed against HLA- and/or non-HLAACKNOWLEDGMLN TSThis work was supported in part by the Dutch Organization for Health Research (TNO),the Dutch Foundation for Medical Research (FUNGO) which IS subsidized by the DutchFoundation for the Advancement of Pure Research (ZWO), and the j Α Cohen Institutefor Radiopathology and Radiation Protection (IRS)We wish to thank Drs Frans Η J Claas and Drs Jan J ν d Poel for valuable suggestionsand Ms Eis Blokland for excellent technical assistance This study could not have beenperformed without the repeated blood donations of the van Rooyen familyREFERENCES1 Biadley BA, Charmot D, Goulmy E, Johnsen HE, Kristensen T, Mawas C, PfefferP, Schendel D, Wank R Histocompatibihty typing by Τ cell mediated lympholysis(CML) Workshop II, technical standardization Tissue Antigens, 16 73, 19802 The European CML Study Group Human histocompatibihty testing by Τ cell-me-diated lympholysis Α European Standard CML technique Tissue Antigens 16 335,19803 Horai S, Claas FHJ, van der Poel JJ, van Rood JJ The outcome of cell mediatedlympholysis JS influenced by the antibiotics in the culture medium Tissue Antigens,1982 (in press)4 Goulmy E, Termijtelen A, Bradley BA, van Rood JJ HLA restnction of non-HLA-A, -B, -C and -D cell mediated lympholysis (CML) Tissue Antigens 8 317, 19765 Bradley BA, Goulmy E, Schreuder GMT, van Rood JJ Target for killer Τ cells InΒ Serrou, and C Rosenfeld, Eds Human Lymphocyte Differentiation Its Applicationto Cancer INSERN Symposium No 8 Elsevier/North-Holland, 1978, ρ 2316 Ben-David A, Orgad S, Danon Y, Michaeli D HLA antigen changes in patientstreated with chloramphemcol Tissue Antigens 3 378, 19737 Majsky A, Chudomel V, Jakoubkova J Effect of chemotherapeutics on lymphocyteHLA antigens in vitro J Immunogenet 2 1<53, 19758 Majsky A, Chudomel V Inactivation of HLA antigens in vitro by action of antibioticsJ Immunogenet 3 29, 19759 Majsky A, Chudomel V, Jakoubkova J Effect of antibiotics and chemotherapeuticson the cytotoxic reaction between HLA antigens and corresponding sera J Immu-nogenet 3 135, 197610 Majsky A, Todorovicova Η The transient loss of HLA antigens on lymphocyte inpatients following admimstration of penicillin and terracychn Foha Haematol 4 502,197811 Scorza-Smeraldi R, Smeraldi E, Fabio G, Bellodi L, Sacchetti F, Rugarli C Interferencebetween anti HLA antibodies and adrenegic receptor-binding drugs Tissue Antigens9 163, 197712 Claas FHJ, Ruma-van Nieuwkoop R, van den Berge W, van Rood JJ Interaction ofpenicillin with HLA-A and -B antigens Hum Immunol, 1982 (in press)

Cytotoxic T lymphocytes directed against HLA-Bw35-linked target determinants show differences in sensitivity towards antibiotics during sensitization period.

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Cytotoxic T lymphocytes directed against HLA-Bw35-linked target determinants show differences in sensitivity towards antibiotics during sensitization period.

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