Immunology 1991 73 44-51

ADONIS 001928059100109E

The effect of transfected MHC class I genes natural killer cells

on

sensitivity to

M. HOLSCHER, A. L. GIVAN* & C. G. BROOKS The Department of Immunology and *The Department of Surgery, The Medical School, Newcastle upon Tyne Acceptedfor publication 31 January 1991

SUMMARY To test the hypothesis that major histocompatibility complex (MHC) molecules protect target cells from lysis by natural killer cells (NKC), we transfected the MHC- B16 melanoma line FO0 with the class I genes encoding Dd, Kb, and Kk. Only low levels of Dd expression could be obtained and there was no protection against NKC. By contrast, Kb and Kk transfectants were obtained which displayed significant resistance to NKC, and with the latter transfectants resistance was clearly related to the level of transgene expression. Various mutants of the F10 line with altered patterns of MHC expression were also obtained. These mutant lines provided evidence that (i) the Db molecule is also capable of inducing resistance to NKC and (ii) high MHC class I expression does not by itself guarantee lowered susceptibility to NKC.

genes were transfected into the cells of a mouse B16 melanoma cell line which fails to express constitutively its endogenous H-2b MHC phenotype.

INTRODUCTION Natural killer cells (NKC) are a class of lymphocyte which can kill certain types of normal and malignant cells. Despite intensive research the factors which control NKC recognition of targets are poorly understood. Although it is clear that target recognition by NKC occurs in a major histocompatibility complex (MHC)-unrestricted manner indeed, many of the best targets lack MHC antigens-there has been tantalizing evidence over the years that genes mapping in or close to the MHC can affect NKC recognition and/or lethal hit delivery. Most spectacularly, the phenomenon of resistance to MHC incompatible allogeneic bone marrow grafts (reviewed in ref. 1) in at least some instances appears to be mediated by NKC,23 as does the closely related phenomenon of rejection of allogeneic lymphocyte grafts.4 In 1985 Kirre pointed out that, in a variety of experimental systems, there seemed to be an inverse correlation between the sensitivity of target cells to NKC and the amounts of MHC antigens the targets expressed.5 Subsequent studies of MHCmutant target cells supported this view.67 These studies can, however, be criticized because of the possibility of co-existing unrelated genetic events. The most direct way of testing the influence of MHC molecules on natural killing is to determine whether increased MHC expression achieved by transfecting cells with MHC genes causes a reduction in NK sensitivity. We describe here a series of experiments in which Dd, Kb, and Kk

MATERIALS AND METHODS

Animals and treatments (CBA x C57BL/6)Fl mice were obtained from Olac (Bicester, Oxon, U.K.). They were injected intraperitoneally with 200 pg poly inosinic: poly cytidylic acid (poly I: C, Sigma, P1530, Poole, Dorset, U.K.) in 200 pl water, and used as donors of splenic NKC 16-18 hr later.

Media, cells and reagents DMEM (074-02100), RPMI-1640 (074-01800) and Hanks' balanced saline solution (HBSS) (076-01200) powdered media were purchased from Gibco (Paisley, Renfrewshire, U.K.) and dissolved in nanopure (Whatman, Maidstone, Kent, U.K.) distilled-deionized water. The DMEM formulation was varied by adding non-essential amino acids (Gibco 043-01140) to final 2 x concentration and 2-mercaptoethanol (Sigma, M3148) at 5 x 10-5 M. RPMI-1640 also received 2-mercaptoethanol at 5 x 10-5 M. HBSS was made up without sodium bicarbonate. Foetal bovine serum (FBS) was purchased from Sigma (F4884) and donor calf serum (DCS) from Gibco (031-06171). The C57 (H-2b)-derived B16 melanoma subline F10 and transfectants were maintained in DMEM supplemented with 10% DCS, 0 25 Mg/ml gentamicin (Gibco 043-05710) and, for transfectants, 1 mg/ml G418 (Gibco 066-1811). Cells were harvested by aspirating culture medium then incubating at room temperature with 0 5 mM EDTA in Ca- and Mg-free Dulbecco's phosphatebuffered saline (PBS) for a few minutes. For induction of MHC

Abbreviations: DCS, donor calf serum; F1, fluorescence index; NKC, natural killer cell; poly I: C, poly inosinic: poly cytidylic acid. Correspondence: Dr C. Brooks, Dept. of Immunology, The Medical School, Newcastle upon Tyne NE2 4HH, U.K.

44

MHC class I genes expression, cells were treated for 3 days with 100 U/ml recombinant mouse interferon-gamma (IFN-y), obtained as a culture supernatant from 21 1A.20 cells, a CHO line transfected with the mouse IFN-y gene,8 kindly provided by Dr A. Morris, University of Warwick, U.K. DNA

The Dd gene cloned into pBR3279 was kindly provided by Dr K. Ozato, National Institutes of Health, Bethesda, MD. The Kb and Kk genes cloned into pBR322'0 were kindly provided by Dr S. Kvist, Ludwig Institute, Stockholm, Sweden. The pBG367 plasmid bearing the Kb gene with the endogenous promoter replaced by the SV40 promoter was kindly donated by Dr A. Mellor, National Institute for Medical Research, London, U.K. The pSV2neo plasmid" was kindly provided by Dr I. Hickson, Nuffield Institute, Oxford, U.K. Plasmids were transformed into JM103 strain of Escherichia coli and DNA prepared by alkali lysis followed by purification on CsCl gradients. 12 Transfection The pSV40neo, pDd, pKb and pKk DNAs were linearized with EcoRl and the pBG367 DNA with Sall followed by precipitation with 70% alcohol and dissolution in sterile nanopure water. Exponentially growing FlO cells were harvested with EDTA, and washed three times in ice-cold phosphate-buffered sucrose (272 mm sucrose, 7 mm sodium phosphate, pH 7.4, 1 mM MgCI2). Cells were resuspended in the same buffer at 5 x 106/ml and aliquots of 2-5 x 106 cells placed in electroporation vials (Biorad, Richmond, CA) together with appropriate linearized DNA (1 ,g/ml pSV2neo +10 Iug/ml MHC class I DNA). They were then pulsed with a Biorad Gene Pulser at 300 V and 25 pFd, giving time constants of about 8A4-10-5. Cells were transferred to plastic tubes, kept on ice for at least 5 min before being diluted with DMEM/10% DCS and added to 100 mm tissue culture grade Petri Dishes (Falcon 3003, Becton Dickinson, Lincoln Park, NJ). After 48 hr, and again every 2-3 days thereafter, cultures were refed with selection medium (DMEM/10% DCS containing 1 mg/ml G418). After about 2 weeks clear colonies had developed (except in control transfectant cultures where cells had been electroplated in the absence of DNA). Individual colonies were picked by micropipette and transferred to the wells of 24-well plates (Costar 3424 Cambridge, MA), and eventually to 25 cm2 flasks (Bibby 251 10B, Stone, Staffs, U.K.). After colony picking, primary transfectant plates were harvested with EDTA and grown up as transfectant lines. All transfected cells were maintained continuously in the presence of 1 mg/ml G418. Approximately 50% of co-transfected colonies were found to express transfected MHC. However, because the level of expression was always low, clones and lines were subjected to several rounds of cell sorting (see below).

Fluorescence staining Cells were washed and resuspended in ice cold HBSS containing 2% FBS and 0-2% sodium azide (H2FA). Aliquots of I x 105 cells were placed in 1 5-ml microfuge tubes in 25 p1 H2FA and 25 Ml medium or monoclonal anti-MHC antibody added at a predetermined saturating concentration. The antibodies used to characterize MHC class I expression on transfectants were: TIB126 (M1/42), rat anti-class I (reacts with most class I

45

molecules but not Db);13 HB 11 (20-8-4S), mouse anti-Kb, Db (but reactivity against Db questionable, see the Results);'4 HB 19 (2811-5S), mouse anti-Db;14 HB25 (16-3-1N), mouse anti-Kk, no cross-reaction on Kb, Db;15 HB27 (28-14-8S), mouse anti-Db;l4 HB41 (28-13-3S), mouse anti-Kb;l4 27-11-13S, mouse anti-Db;I4 H97, mouse anti-Dd, no cross-reaction on Kb, Db;16 and S19-8 mouse

anti-C57-fi2-microglobulin (anti-,f2mb).I7

After 20 min on ice, cells were washed twice with 1 ml of H2FA and microfuged for 20 seconds at 15,000 g. They were resuspended in 25 p1 H2FA and 25 p1 FITC-conjugated goat anti-rat Ig or goat anti-mouse Ig (CALTAG, San Francisco, CA) added at a predetermined saturating concentration. After 20 min on ice, cells were washed once, resuspended in 0 5 ml H2FA and propidium iodide (PI) added to 2 Mg/ml. Cells were analysed on a Becton-Dickinson FACS 420 with four parameters, non-viable cells being excluded by a combination of forward and right angle scatter and PI fluorescence. Histograms were generated by plotting cell number versus logl0 fluorescence intensity at 530 nm. Median fluorescence values were determined for control and stained cells and the fluorescence index (FI) for each cell/antibody combination calculated as: FT = median fluorescence intensity of cells stained with first layer + second layer median fluorescence intensity of control cells stained with second layer alone

A Fl of 1 0 represents no staining. Cell sorting

Transfectant lines were stained with appropriate anti-class I antibodies using a scaled-up version of the procedure described above, but using sterile H2F lacking sodium azide. The 5% most strongly fluorescent cells were isolated by flow cytometry, expanded in culture for about 1 week, then sorted again in the same manner. Up to seven rounds of sorting were performed, giving a substantial increase in mean cellular expression of transfected MHC. After the last round of sorting, cells were usually cloned by adding approximately 100 cells to a 100-mm Petri dish and picking colonies 10-14 days later.

Cytotoxicity assay Target cells were labelled by incubating approximately 2 x 106 cells for 1 hr at 370 in 200 pl of saline containing 50% FBS and 100 OCi Na2 5"CrO4. Cells were washed twice with 10 ml HBSS/ 1% FBS and resuspended at 5 x 104/ml in RPMI-1640/5% FBS (RSF). Spleen cells from poly I: C-treated mice were freed of red cells by water lysis by resuspending in about 200 Ml medium, and mixing with 5 ml of water. After about 10 seconds, 5 ml 2 x concentrated PBS were mixed in, cells spun down, and suspended at 5 x 106/ml in R5F. Serial 100 pl dilutions of this suspension were made in V-bottomed microtest plates (Nunc 245128, Roskilde, Denmark) and 100 pl of target cell suspension added, giving effector: target cell ratios of 100: 1, 50: 1, 25: 1 and 12-5:1. Wells lacking spleen cells served to measure spontaneous 51Cr release. After 4 hr at 370, 100-pl aliquots of supernatant medium were counted on a gamma-spectrometer. Percentage cytotoxicity was calculated as 100 x (E - S/100 - S), where E = percentage 5'Cr release in the presence of effector cells, and S = percentage 5'Cr release in the absence of effector cells.

46

M. Holscher, A. L. Givan & C. G. Brooks -50

20a)

-25

0

a)1 u U

0

(quantified as described below) and the level of expression of MHC class I antigens. With 1 unit/ml IFN the effect on both parameters is clear, with 0 1 U/ml IFN it is just detectable, and with 100 U/ml IFN it is maximal. These results show that the FlO line behaves in the typical manner described by Kdrre, and that if MHC class I molecules are responsible for IFN-induced resistance to NKC then sub-maximal levels of class I expression are sufficient to reduce killing significantly.

-0.0

10-1

0

10° IFN (U/ml)

102

10'

Figure 1. The effect of IFN treatment of F10 cells on MHC class I expression and susceptibility to NK lysis. Cytotoxicity was quantified from the slopes of regression lines as illustrated in Fig. 2. The results were averaged from two individual experiments, giving similar results.

20-~~~~~ 20

1

04

/

0

0

6 12 25 Effector cells/well (x

50

10-4)

Figure 2. NKC killing of F10 transfectants. Percentage cytotoxicity is plotted as a linear function of effector cell number, together with the associated regression lines. Targets were: control transfectant line 32 without IFN treatment (0), control transfectant line 32 with IFN treatment (0) Dd transfectant line B21.1S5.35 without IFN (01), and Dd transfectant line B21.1S5.35 with IFN treatment (-). RESULTS

General experimental design Three days prior to testing cell lines for NK sensitivity, they were subcultured into two flasks, to one of which was added 100 U/ml rIFN-y. Cells were then harvested with EDTA, labelled with 5'Cr, and set up in a 4 hr cytotoxicity assay with various numbers of spleen cells from poly I: C-treated (CBA x C57)Fj mice. The unused 5'Cr-labelled cells were then stained with anti-MHC antibodies and fluorescence quantified by flow cytometry. The F10 melanoma shows an inverse correlation between MHC expression and sensitivity to NKC Detailed studies with a panel of anti-class I antibodies and with the S19-8 anti-32M antibody, which would presumably react with any classical or non-classical class I molecules, failed to reveal any class I expression on untreated F10 cells. Figure 1 shows the results of treating F IO cells with various doses of IFN. There is a marked inverse correlation between NK sensitivity

Dd-transfectants Figure 2 illustrates the results from one experiment in which the NK sensitivity of various cell lines was measured. As described previously,'8 at values of percentage cytotoxicity less than about 25% there is a linear relationship between percentage cytotoxicity and effector cell number. Consequently, the relative NK sensitivity of cell lines can be determined by comparing the slopes and standard deviations of regression lines fitted through the origin. The full results of the experiment in Fig. 2 are documented in Table 1. Following culture with IFN substantial amounts of class I Kb and Db locus products were expressed by FIO and its NK sensitivity fell to less than half. Clone 32 was obtained from a cotransfection of FI 0 with the neo gene + Dd gene. Although it clearly expressed the neomycin-resistance phenotype, it expressed no detectable constitutive or inducible Dd. It was therefore used as a neo-transfected control line. Like the parental FlO line, its NK sensitivity was markedly reduced by IFN pretreatment. B21.1S5.35 is a Dd-expressing transfectant clone. In the absence of IFN treatment it expressed Dd molecules but not endogenous Kb or Db. Despite this constitutive class I MHC antigen expression it showed no reduction in NK sensitivity compared to neo control or parental cell lines; in fact, in this and several other experiments it displayed a slightly increased NK sensitivity. Following treatment with IFN there was a huge increase in transgene expression, induction of endogenous H-2b class I molecules, and loss of NK sensitivity. Essentially identical results were obtained with a second Dd expressing clone isolated at the same time (not shown). Clone 27 was isolated from the original transfection mixture at the same time as clone 32. Detailed analysis, however, showed it to be a mutant which, as judged by lack of reactivity with H97 anti-Dd or HBl 1 anti-Kb/Db antibodies, lacked MHC expression even after IFN treatment. Nonetheless, IFN treatment caused a similar degree of reduction of NK sensitivity to that seen

in control cell lines.

Superficially, these results contradicted the hypothesis of Karre in two ways: (i) MHC class I-expressing transfectants did not have reduced NK sensitivity and (ii) IFN-mediated reduction in NK sensitivity could occur without MHC expression. Both conclusions, however, require substantial qualification. Firstly, although the HB 1 antibody used to establish lack of Kb/Db expression on clone 27 after IFN treatment has been shown in congenic recombinant strains to react strongly against both K locus and D locus molecules,'4 a finding reproduced in our laboratory using appropriate congenic strains, three other Db-reactive monoclonal antibodies (HB 19, HB27, and 27-1113S) were later found to react strongly with IFN-treated clone 27 cells; two Kb-reactive antibodies (TIB 126, HB41) did not react. It seems likely, therefore, that clone 27 was a mutant

47

MHC class I genes Table 1. NK sensitivity of Dd transfectants MHC expressiont

Relative NK sensitivity (%) Line

Transfected with

IFN treatment

H97 anti-Dd

HBl 1 anti-Kb/Db

FIO

0

+ _ + + -

10 1-0 10 1-0

09 6-2

1-0

3-43 +0-11

39

2-06+0-24

19

1.0

4-98+0-24

25-4 10 1-0

2-8

1-44+0 07

1.0 1.0

4-26+0-07

32

neo + Dd

B21.1S5.35

neo+Dd

27

neo+Dd

+

NK

sensitivity:

-IFN§

+ IFNT

2-88+0-02 41***

1-19+0-11

1-61 +0-14

60**

145** 29***

124** 38***

t Figures show the fluorescence index calculated as described in the Materials and Methods (1 0 =no MHC expression). I Figures show slopes of regression lines + SE, calculated as in reference 18. § NK sensitivity of test line expressed as a percentage of control neo-transfectant. Statistical significance was calculated by Student's t-test: *P