Hum Genet (1987) 76:369-374

© Springer-Verlag 1987

Unsuitability of the assay for cell-mediated lympholysis in inbred mice for H-Y antigen determination of human cells

Andreas Braun and Hartwig Cleve

Institut far Anthropologie und Humangenetik der Universitfit, Richard-Wagner-Strasse 10/I, D-8000 Mtinchen 2, Federal Republic of Germany

Summary. This study examined the H-Y-specific in vitro re- stimulation of splenocytes from in vivo intraperitoneaUy (i.p.) primed C57B1/6 (B6) female mice. In vivo priming was car- ried out with human male or female fibroblasts or peripheral blood lymphocytes, respectively. It was attempted to measure the in vitro H-Y-specific activity by cell-mediated lympholysis and by cell proliferation. S[H]Thymidine incorporation was determined in mixed lymphocyte cultures (MLCs) of xeno- geneic primed female splenocytes (responder cells) and of syngeneic lethally irradiated male splenocytes (stimulator cells). The xenogeneic H-Y presentation by in vivo sensitiza- tion did not induce in the in vitro restimulation system an H-Y-specific cell proliferation or in the effector phase the gen- eration of H-Y-specific killer cells. The assay for cell- mediated lympholysis and lymphocyte proliferation after xenogeneic priming and syngeneic in vitro restimulation is, thus, not suitable for H-Y testing of human cells.

Introduction

The existence of a male-specific cell surface antigen was first demonstrated by Eichwald and Silmser (1955). Grafting ex- periments showed that in inbred strains of mice the females rejected syngeneic male skin. This finding was ascribed to the presence of a histocompatibility antigen specific for male cells which was named the H-Y antigen. The genetic difference be- tween female and male inbred mice is, apart from the absence of one X-chromosome, the presence of the Y-chromosome in males. This fact supports the hypothesis that the genetic con- trol elements (regulatory and/or structural gene(s)) for the H-Y antigen may be located on the Y-chromosome (Billing- ham and Silvers 1960). In a review, Gasser and Silvers note in 1972 that only female mice of the H-2b haplotype are capable of rejecting in any regular manner isogeneic male skin grafts. The in vitro demonstration of cytotoxic T-lymphocytes (CTLs) against the H-Y antigen was also restricted to the H-2b haplotype (Goldberg et al. 1973; Gordon et al. 1975): only in females with the H-2b haplotype were cytotoxic T-lym- phocytes generated against syngeneic male target cells.

It was subsequently demonstrated that also in other as H-2b haplotype mice responsiveness against the H-Y antigen may exist: this is caused by gene complementation in F1 off-

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spring from crosses of non-responder inbred strains (Simpson and Gordon 1977; Matsunaga and Simpson 1978) or by non- H-2-1inked genes for a positive response (Fierz et al. 1982a, b). However, also by changing the route of immunization (intraperitoneal versus footpad), Mfillbacher and Brenan (1980) induced the generation of CTLs in a secondary MLC from so called non-responder mice, that is CBA. At last, Juretid et al. (1985) demonstrated that two non-responder mice can be supplemented: B10.A(4R) used as male donor for the in vivo priming of female B10.A(2R) can induce helper effects for the responsiveness against the H-Y antigen in the recipient.

Concerning the H-2b haplotype, the immune response against the H-Y antigen is restricted to a defined class I anti- gen and to certain class II antigens. The H-Y-specific CTLs of the H-2b haplotype are restricted to the right-hand side of the H-2 gene complex, that is to H-2Db (Gordon et al. 1975). For the helper/effector cell interactions two dominant class II im- mune response genes have been distinguished. One is mapped to the IAb region and responsible for the generation of H-Y- specific cytotoxic T-lymphocytes (Hnrme et al. 1978a; B6h- met von et al. 1979; Waal de et al. 1983a,b). The other was observed by Hurme et al. (1978b) and found to be responsible for the rejection of syngeneic male skin grafts. This immune response gene was originally ascribed to the IB region. The existence of this putative IB region has recently come into question (Simpson et al. 1986a): DNA analysis of the segment of the MHC region coding for the various alpha- and beta- chains of the A and E antigens appears to exclude the possibil- ity that other class II antigens could be mapped between A and E. The nature of the restriction element IB as cause for the H-Y-dependent transplant rejection is a matter of con- troversy (Simpson et al. 1986a; Klein 1986; Simpson et al. 1986b). Conceivable are a special type of interaction between T-helper and T-suppressor cells (Klein 1986) or the presence of unusual class II antigens as a result of new combinations of the A and/or E molecules (Simpson et al. 1986a). Therefore, we will refer to this putative restriction element as "IB".

For generation of H-2-restricted H-Y-specific CTLs in females of the inbred strain C57B1/6 (B6) syngeneic male cells are used for the in vivo priming through intraperitoneal injec- tion and also for the subsequent in vitro restimulation. HoW- ever, also sensitization in vivo with allogeneic skin grafts fol- lowed by challenge in vitro with syngeneic male cells was suc- cessful: cytotoxic T cells against the H-Y antigen were gener- ated (Gordon et al. 1976). On the other hand, after xeno-

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geneic in vivo priming by intraperitoneal injection with male cells the primed B6 females rejected syngeueic male skin grafts in a second set manner (Silvers and Yang 1973; Wiberg 1985; Wiberg and Gfinther 1985; Wiberg and Fredga 1985). In an independent study, Worley et al. (1985) observed that a male-specific sensitization of B6 females with xenogeneic (rat and human) cells does not occur; they concluded that a xeno- geneic reaction was not useful for the classification of H-Y positive or negative cells.

In this study, we attempted to find a cellular in vitro system for the determination of the H-Y antigen on human cells. Therefore, we examined whether priming in vivo with xeno- geneic male (or control female) cells by intraperitoneal injec- tion of C57Bll6 female mice followed by restimulation in vitro with syngeneic male cells can be used for the demonstration of an H-Y-specific reaction. The xenogeneic cells which we applied were human fibroblasts and unstimulated, and PHA stimulated human peripheral blood lymphocytes. After syn- geneic restimulation we attempted to measure the generation of H-Y-specific cytotoxic T-lymphocytes as well as the H-Y- specific proliferation of female cells. The advantages in com- parison with transplantation experiments (Wiberg 1985) would be that the estimation by an in vitro assay is based on measurements for [51Cr]release or [3H]thymidine incorpora- tion. This test is, thus, independent from expertise for trans- plantation experiments and the observation of transplant re- jections. Furthermore, the results are obtained in a shorter time.

Materials and methods

Animals

Female and male C57B1/6 (B6) mice at an age of 3 months were obtained from Charles River GmbH, Sulzfeld, FRG. Male CBA mice were purchased from SAVO-Ivanovas, med. Versuchstierzuchten GmbH, KiBlegg, FRG.

Human fibroblasts

All fibroblasts were karyotyped in our zytogenetic laboratory. The following cell lines were available for in vivo priming: F15 (gonosomes: XX/age at the time of biopsy: 7 years); F40 (XX/ 26); F31 (XY/4); F13 (XY/fetal); F6 (XY/fetal); F48 (XO/17). All fibroblast lines were established from skin biopsies.

Cell cultures

Fibroblasts were cultured in T75 Nunclon tissue culture flasks at 37°C and 5% CO2. The culture medium was DMEM (Gib- co) supplemented with 20raM glutamine, 15% fetal calf serum (FCS) (Boehringer) and penicillin/streptomycin (Sero- med 1 ml/i00 ml).

The PHA stimulation medium for PBLs was RPMI 1640 (Gibco) supplemented with 10mM glutamine, 10% FCS, penicillin/streptomycin (Seromed lml/100 ml), 10 mM N-2- hydroxyethylpiperazine-N'-2-ethane sulphonic acid (HEPES) and 5 gg/ml PHA (Wellcome). The cell concentration was 1 × 106/ml at the beginning of stimulation. Incubation was for 72h at 37°C and 5% CO2.

Pure splenocyte suspensions of mice for responder, stimulator, or target cells were prepared by centrifuging the whole splenocyte suspension over a Percoll (Pharmacia) gra- dient with the density of 1.088 g/ml. In the interphase was a maximal impurity of 5% erythrocytes for the splenocyte prep- arations.

Mixed lymphocyte cultures were performed in T25 Nunclon tissue culture flasks standing upright. The medium was RPMI 1640 supplemented with 10mM glutamine, 5% FCS, penicillin/streptomycin (Seromed 1 ml/100ml), 10mM HEPES, and 0.05 mM 2-mercaptoethanol. Cell concentration per millilitre was 5 x 106 responder cells (primed B6 female splenocytes) and 5 x 106 stimulator cells (B6 male splenocytes irradiated with 2200 R). The total volume per MLC was 12- 14ml. The incubation was at 37°C and 5% CO2 for 5 days.

Target cells in the cell-mediated lympholysis (CML) assay were female and male B6 splenocytes, which were stimulated for 72h at 37°C and 5% COB in MLC medium supplemented with 5 gg concanavalin A (con A) (Difco)/ml. Cell concentra- tion was 1 x 106/ml at the beginning of the stimulation.

Cell-mediated lympholysis assay

Con A stimulated target cells (5 x 106) were incubated in 0.2 ml medium with 125 gCi sodium 51chromate (New England Nuclear). After 60-90 min the target cells were centrifuged over Ficoll Paque and the interphase was washed twice in medium. In U-shape 96 well microtiter plates (Greiner) vari- ous concentrations of attacker (5-day-old MLC suspension) and target cells were incubated in triplicates for 3 h. After this time the supernatant was measured for released 51Cr in a gamma counter (Packard). Specific 51Cr release was calcu- lated as follows:

Human peripheral blood lymphocytes

Peripheral blood lymphocytes were isolated from heparinized blood of normal male (three) and female (three) donors by Ficoll Paque (Pharmacia) density gradient centrifugation.

Priming

Either 1 x 107 syngeneic male splenocytes or 1-2 x 106 fibro- blasts or 5 x 106 unstimulated PBLs or 2 x 106 phytohaem- agglutinin(PHA)-stimulated lymphocytes were used for intra- peritoneal priming of 3= to 4-month-old B6 female mice. Un- stimulated and PHA stimulated human lymphocytes were ir- radiated (2200 R) before priming. Mixed lymphocyte cultures or proliferation assays were performed 2-5 weeks after prim- ing.

% CML = Experimental cpm - spontaneous cpm x 100 Maximum cpm - spontaneous cpm

In experiments with spontaneous cytolysis between 20% and 30% this calculation procedure may lead to negative values for undestroyed target cells (see Figs. 1,2).

Cell proliferation assay

Cell proliferation assay was performed in U-shape 96 well microtiter plates (Nunc). The medium was the MLC medium with the exception of 10% FCS. Various responder and stimulator cell concentrations (see Results) were given to- gether in 0.2ml medium and incubated at 37°C and 5% COB for 24, 48, 72, and 96h. Six hours before harvesting 2gCi

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[3H]thymidine (New England Nuclear) was added per well. The DNA with incorporated [3H]thymidine was harvested and washed onto filter paper (Dunn Labortechnik) by means of an automated cell harvester. All tests were made in tripli- cate and the standard deviation was calculated.

Results

The generation of cytotoxic T-lymphocytes against the H-Y antigen with the assay described by Gordon et al. (1975) is highly reproducible. Figure 1 shows a strong H-Y-specific ac- tivity of a secondary MLC from B6 females primed in vivo with syngeneic male cells. Only male but not female cells were lysed. We performed this assay as a positive control when we tested B6 females, who were primed in vivo with xenogeneic human male and female cells. The individual activity of four positive controls ranged between 77.3% and 33.0% specific cell-mediated lysis at an attacker:target ratio of 100:1. The spontaneous SlCr release of the target cells at all assays was between 15% and 30% of the maximum.

The macroscopic appearance of the spleens from xenogeneic primed B6 females was different from those who were unprimed or primed with syngeneic cells. The spleens were larger, not so compact, and darker; therefore, we con- cluded that the immunizations with xenogeneic cells had been followed by a reaction. However, in the secondary mixed lym- phocyte cultures there was no H-Y-specific activity (see Fig. 2). For each cell line the in vivo priming and the in vitro restimulation was performed 2 or 3 times, respectively. There-

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Fig.3A-D. Influence of cell concentrations and kinetics for the H-Y-specific cell proliferation in MLCs. Responder cells were splenocytes from a B6 female primed in vivo with syngeneic male cells. The MLCs were started 3 weeks after priming. In the MLCs were either no stimulator cells (A) or irradiated syngeneic male stimulator cells (.) or irradiated syngeneic female stimulator ceils (©). The responder: stimulator ratio was 1 : 1. The counts for responder cells per well were 50000 in A, 100000 in B, 150000 in C, and 200000 in D

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Fig.4. Cell proliferation in mixed lymphocyte cultures (MLCs) from responder cells of B6 females primed in vivo with human fibroblasts and challenged in vitro with irradiated syngeneic splenocytes from either males or females. For F6, F40, and F48, MLCs were started 5 weeks and for F13 and F31 2 weeks after priming. The positive control was performed 3 weeks after priming. Responder cell concentrations were 200000 cells/well and the [3H] thymidine incorporation was mea- sured at 72 h after the beginning of the MLCs

fore, the result is clearly negative: there is no generation of H-Y-specific CTLs after xenogeneic in vivo priming of B6 females with either human fibroblasts or human, unstimulated or PHA-stimulated lymphocytes.

As a further control the CML after priming and restimula- tion was examined within the same species with the use of al- logeneic cells for in vivo priming. Male splenocytes from CBA mice were applied i.p. to B6 female mice for in vivo priming. In vitro restimulation of splenocytes from these pretreated female mice with syngeneic male cells did not induce the pro- duction of cytotoxic killer cells capable of recognizing specifi- cally male target cells (results not demonstrated).

We examined, furthermore, the possibility that an H-Y- specific reaction in the MLC was induced other than the lysis of target cells as mediated by cytotoxic T-lymphocytes. It has been shown (Liew and Simpson 1980) that the H-Y-specific rejection of skin grafts is not transferred by CTLs but by a T-cell subset associated with the delayed-type hypersensitivity (DTH) response. In this case the induction of an H-Y-specific activity could be missed if only the generation of killer cells in the cell-mediated lysis assay was monitored. Therefore, we in- cluded the cell proliferation assay and measured the incorpo- ration of [3H]thymidine into DNA. To establish the assay in our laboratory, we determined first the appropriate cell con- centrations and observed the kinetics of the proliferation assay in the syngeneic system (C57B1/6). Figure 3 shows these results. The responder : stimulator ratio was 1 : 1. At low cell concentrations per well in the microtiter plate (50000) cell proliferation could not be observed; there was no measurable difference between secondary unstimulated and stimulated cells over a period from 24 to 96h. At 100000 cells/well there was a low specific difference at 48 h and later. By using 150000 or 200000 cells/well a clear-cut difference can be observed which is most apparent at 72 h after the beginning of the MLC. Different responder: stimulator ratios, i.e. 1 : 1, 1 : 2, and 1 : 3, do not result in a more pronounced difference. Therefore, we measured the activity of the secondary MLCs at 200000 ceils/ well and after 48h and 72h. Comparing different sets of MLCs, there were individual differences in [3H]thymidine in- corporation values (Fig.4). The H-Y-specific proliferation was only seen in the positive controls, which were B6 female mice primed in vivo and restimulated in vitro with syngeneic male splenocytes. These were associated with the positive re- suits in the CML assays. However, the use of the proliferation assay did not disclose an H-Y-specific activity in the MLCs, when B6 females had been primed with human fibroblasts and were restimulated in vitro with syngeneic female (control) or

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male splenocytes. All tests were performed at least twice; the results were uniformly negative.

Discussion

Wiberg et al. (Wiberg 1985; Wiberg and Gfinther 1985; Wi- berg and Fredga 1985) demonstrated recently after priming in vivo of B6 female mice with xenogeneic male or H-Y-positive cells from humans or wood lemmings accelerated rejection of syngeneic male skin transplants in a second set manner. It was our aim to replace the transplant rejection system by the mea- surement of cell-mediated lympholysis and/or cell prolifera- tion. However, our results are clearly negative concerning the xenogeneic H-Y-specific i.p. in vivo priming. In splenocyte preparations from such primed B6 female mice we have not detected memory T cells against the H-Y antigen in secondary MLCs, neither with the use of the CML assay nor with the help of the cell proliferation assay. In the studies on the cellu- lar rejection for the H-Y antigen reported, thus far, two differ- ent routes for the allogeneic and xenogeneic in vivo priming have been used.

In the allogeneic in vivo priming experiments described by Gordon et al. (1976) skin grafting is used as the primary sen- sitization method. The positive results following the subse- quent syngeneic graftings or syngeneic secondary MLCs show that the allogeneic H-Y antigen will be processed from the female macrophages and presumably presented in the context with IAb and "IBb" antigens. As a result thereof, T-helper cells ("IBb") are induced for the graft rejection mediated by DTH cells in vivo (Liew and Simpson 1980) as well as T- helper cells (IAb) for the T-cell-dependent cytotoxic reaction mediated by CTLs in vitro.

In the xenogeneic in vivo priming experiments (Silvers and Yang 1973; Wiberg 1985; Wiberg and Gfinther 1985; Wiberg and Fredga 1985) intraperitoneal cell injections were employed as the primary sensitization method. Skin grafting was used in the secondary reaction as the evaluation method with positive results.

In our experiments, xenogeneic i.p. priming did not lead to positive results when the secondary response was assessed in a restimulation assay in vitro. It is conceivable that the female macrophages process during i.p. in vivo sensitization the xenogeneic H-Y antigen only in the context of the "IBb" anti- gen. We deduce from these observations that in vivo priming by skin transplants stimulates both T-helper subpopulations, the "IBb"-dependent as well as the IAb-dependent cell sets, whereas i.p. in vivo priming stimulates the "IBb"-dependent cells only. In agreement with this interpretation was our ex- periment with allogeneic male cells (CBA, H-2k) for i.p. in vivo priming of B6 female mice. Also in this experiment we did not observe any H-Y-specific activity in vitro. It excludes, furthermore, that our negative results were specific for xenogeneic priming only.

The failure to obtain an H-Y-specific activity in the cell proliferation assay may be explained by the recent findings of Biasi et al. (1985): the H-Y antigen will not be presented in vitro (MLC) by female macrophages in the responder cell sus- pension but by male macrophages in the stimulator cell sus- pension, i.e. the H-Y antigen will not be processed in vitro by female macrophages. If one assumes that the male macro- phages do only present the H-Y antigen in the context of IAb and not of "IBb"; our negative results would be expected,

since in the responder cell suspension there are not IAb-de- pendent memory T cells, only those which are "IBb" depen- dent. The female responder cells in the in vitro restimulation system thus fail to recognize the H-Y antigen of the male stimulator cells.

Xenogeneic sensitization with human cells and transplant rejection was also examined recently for H-Y antigen recogni- tion by Worley et al. (1985). They did not observe significant and reproducible acceleration of transplant rejection. They used white blood cells for in vivo i.p. priming of B6 female mice instead of the fibroblasts employed by Wiberg (1985). Furthermore, Worley et al. (1985) performed grafting 8 days after priming in contrast to Wiberg (1985), who grafted 30-34 days later. Also the type and the size of the grafts may have been different in the two transplantation studies. These differ- ences in the in vivo priming and evaluation procedures may explain the divergence of the results of the two laboratories. The divergence of their results, however, also points to the difficulties in establishing on this basis a practical typing meth- od for H-Y antigen determination on human cells. At the pre- sent time, also the "IBb"-dependent DTH cannot be mea- sured in mice objectively with a sufficient degree of precision in order to be useful for an H-Y antigen assay of xenogeneic cells.

H-Y antigen determination in vitro on human cells in a re- liable and objective manner was made with human female cytotoxic T-lymphocytes against human mate target cells (Goulmy et al. 1980; Pfeffer et al. 1981; Goulmy et al. 1983). However, the disadvantage of this test system was its MHC re- striction: only such human cells were typable for the H-Y anti- gen which were positive for HLA-A2 and/or HLA-B7. The assay depends on the availability of cytotoxic T-lymphocytes from female patients who had received multiple blood transfu- sions and a bone marrow transplant or a kidney transplant, re- spectively. A reliable H-Y antigen determination procedure which is generally applicable and available as a diagnostic tool is, therefore, still an unresolved problem.

In analogy to the allogeneic H-Y test system from Gordon et al. (1976) it is conceivable that determination of the H-Y transplantation antigen on human cells can be made by in vivo priming of B6 female mice with human skin transplants and subsequent measurement of T-cell stimulation or generation of cytotoxic T-lymphocytes following in vitro restimulation with syngeneic male cells.

Acknowledgements. This study was supported by grants from the Deutsche Forschungsgemeinschaft (C1 27/11-1-3 and C1 27/13), for which we are grateful. We would like to thank Professor Dolores J. Schendel, Department of Immunology, University of Munich, for her discussions, encouragement, and help with the MLCs. We thank Ms. A. Brandhofer for her assistance with the tissue culture work. Dr. J. Wienberg, Ms. U. Maurer, and Ms. H. Lieb performed the cyto- genetic studies, which is gratefully recorded.

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Received January 7, 1987 / Revised February 11, 1987