Elicitation of primary anti-Sendai virus cytotoxic T lymphocytes with purified viral glycoproteins

Eur. J . Immunol. 1980.10: 923-928 Elicitation of anti-viral cytotoxic T lymphocytes 923

25 Goidl, E. A , , Romano, T. J . , Siskind, G . W. and Thorbecke, G. J., Cell. Immunol. 1978. 35: 231.

26 Goidl, E. A , , Innes, J . B. and Weksler, M. E., J . Exp. Med. 1976. 144: 1037.

27 Weksler, M. E., DeKruyff, R., Dobken, J . and Siskind, G. W., in Segre, D. and Smith, L. (Eds.), Immunological Aspects of Aging, Elsevier Publishing Co., Amsterdam, in press.

28 Dobken, J., Weksler, M. E. and Siskind, G . W., Cell. Zmmurzot.,

29 DeKruyff, R. H., Rinnooy Kan, E . A., Weksler, M. E. and Sis-

30 DeKruyff, R. H., Kim, Y. T., Siskind, G. W. and Weksler, M. E.,

in press.

kind, G . W., Cell. Immunol., in press.

J . Immunol., in press.

Maria McGee, Arthur H. Hale and Marguerite Panetti

Department of Microbiology and Immunology, The Bowman Gray School of Medicine of Wake Forest University, Winston-Salem

Elicitation of primary anti-Sendai virus cytotoxic T lymphocytes with purified viral glycoproteins*

The requirement of integration of viral proteins into cell surface membranes of host cells for elicitation of anti-Sendai virus (SV) cytotoxic T lymphocytes (CTL) has been investigated. The purified hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of SV incorporated into phospholipid vesicles was used to analyze this question. Phospholipid vesicles possessing an active HN and F glycoprotein were capable of eliciting both anti-SV CTL and antibodies. However, the incorporation of an inactive F glycoprotein into HN-containing vesicles or its absence from such vesicles resulted in stimulation of only anti-SV antibodies and not anti-SV CTL.

1 Introduction

An important question in viral immunology is the mechanism(s) by which anti-viral cytotoxic T lymphocytes (CTL) are elicited in viva. The suggestion that elicitation of CTL is dependent upon the dual recognition of the viral antigen and gene products of the major histocompatibility complex (MHC) makes the understanding of the mechanism of elicitation of anti-viral CTL a complex problem. Infection of the host with infectious virus particles results in the synthesis of virus- specific molecules which may be expressed on the surface of infected cells. These virus-specific cell surface antigens may then be presented to the immune system in the context of the H-2K and H-2D gene products, thus resulting in the elicitation of anti-viral CTL.

Many viruses, when injected into an animal in an uninfectious form, do not elicit CTL ([l, 21 and Hale, unpublished results). Other inactivated viruses, however, have been shown to elicit

[I 28601

* This work was supported by a grant to the Bowman Gray School of Medicine, Cancer Center Support (CORE) grant (2-P30-CA

This work was also supported by the American Cancer Society, MV-41 and an NIH grant, A1 15785.

12197-08).

Correspondence: Arthur H. Hale, Department of Microbiology and Immunology, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC 27103, USA

Abbreviations: CTL: Cytotoxic T lymphocytes DC: Sodium deoxycholate E : T ratio: Effector : target cell ratio F: Active fusion (protein) Fo: Inactive fusion (protein) HN: Hemagglutinin-neuraminidase MHC: Major histocompatibility complex PAGE: Polyacrylamide gel electrophoresis PBS: Phosphate-buffered saline SV: Sendai virus SDS: Sodium dodecyl sulfate C: Complement MDBK: Madin-Darby bovine kidney (cells)

0 Verlag Chemie, GmbH, D-6940 Weinheim, 1980

both primary and secondary anti-viral CTL [3-51. A hypothesis to explain these differences in the ability of different inactivated viruses to elicit anti-viral CTL is that those inactive viruses capable of eliciting anti-viral CTL have the capacity to fuse with host cells and to render the host cells capable of stimulating an anti-viral CTL response.

Recently, Gething et al. [6] demonstrated that the process of fusion was probably important for the ability of UV-inactivated Sendai virus (SV) to render target cells susceptible to lysis by anti-SV CTL. SV grown in Madin-Darby bovine kidney cells (MDBK) possesses an inactive fusion protein (Fo) and is therefore unable to fuse with the plasma membrane of cells. The hemagglutinin-neuraminidase (HN) glycoprotein of this inactive virus is completely active [7, 81. Induction of fusion activity requires a proteolytic cleavage of Fo, which occurs when SV is grown in embryonated eggs. MDBK-grown SV can be treated in vitro with low concentrations of trypsin. This treatment causes full restoration of the fusion and hemolytic activity by converting the Fo into the active form (F). Gething et al. [6] utilized this system by testing the ability of MDBK-grown SV (Fo) and MDBK-grown SV treated with trypsin (F) to render targets susceptible to lysis by anti-SV CTL. The results indicated that, indeed, only SV with F was capable of modifying target cells in such a way as to render them susceptible to lysis by anti-SV CTL. We have confirmed these experiments and extended them to show that F incorporated into liposomes containing an active HN was also capable of eliciting secondary anti-SV CTL. The presence of Fo incorporated into HN-containing liposomes reduced the ability of these liposomes to elicit secondary anti-SV CTL [7]. This differential capacity of liposomes containing either F or Fo and HN to elicit anti-SV CTL was quantitative; i.e. high concentrations of liposomes containing HN and Fo could elicit secondary anti-SV CTL. In this study, we have evaluated the ability of UV-inactivated SV with or without F and artificial phospholipid vesicles containing purified HN, F, HNF or HNFo to

0014-29 80/80/12 12-0923$O2.50/0

924 M. McGee, A. H. Hale and M. Panetti Eur. J. Imrnunol. 1980.10: 923-928

elicit primary in vivo anti-SV CTL. The results indicate that both active HN and F incorporated into the same lipid vesicle is needed for effective elicitation of anti-SV CTL. Elicitation of anti-SV antibodies, however, did not show this dependence and could be elicited with noninfectious SV possessing F or Fo or with liposomes containing purified HN, F, HNF or HNFo glycoproteins.

2 Materials and methods

2.1 Mice

Male and female mice (8-10 weeks old) of the following strains were used: BALB/c (d, d), BALB.B (b, b) and (BALB/ c X BALB . B)F,. These were either purchased from Cumber- land View Farms (Clinton, TN) or produced in our own breed- ing colony. Mice were tested before use for anti-SV antibodies by inhibition of hemagglutination (see Sect. 2.7). Only animals found to be negative were used for elicitation.of primary anti- SV CTL. Although the animals used were judged naive, we of course cannot rule out the possibility of a previous infection.

2.2 Virus

SV with F glycoprotein was grown in MDBK, purified and activated by treatment with trypsin (5 pgiml, TPCK-trypsin, Worthington, Freehold, NJ) as described by Scheid and Chop- pin [S]. This virus was inactivated by UV light, as described previously 17, 91. SV grown in MDBK cells and inactivated by UV light will be referred to as SV-MDBK (Fo glycoprotein). SV grown in MDBK cells, inactivated by UV light and incubated with trypsin, will be referred to as SV-T (F glycoprotein).

2.3 Purification of SV glycoproteins

The glycoproteins of MDBK-grown SV with and without trypsin treatment (see Sect. 2.2) were isolated by extraction of the purified virus with Triton X-100 (final concentration of 2.0%) in 1 M KCI, and the M protein was removed from the extract as described by Scheid and Choppin [8]. The Triton X-100 was removed by exchange with sodium deoxycholate (DC) by 2 cycles of chromatography on Sephadex G-75 in 10 mM sodium phosphate, 0.9% NaCI, 0.2% DC, pH 7.4. HNF is the purified mixture of HN and F. HNFo is the purified mixture of HN and Fo. Purity was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [lo]; only HN and F or Fo glycoproteins were observed on these gels (data not shown and [7]).

For purification of the individual F and HN glycoproteins, a different procedure was followed. Purified SV grown in embryonated eggs was extracted with 0.25% Triton X-100 in 0.01 M sodium phosphate buffer, pH 7.2, at 6.4 mg protein/ml for 30 min at room temperature with repeated mixing in a homogenizer. HNF was the supernatant of the extracted proteins after centrifugation at 200000 x g for 1 h [7]. The yield of HN was 20% as measured by neuraminidase enzyme assay. HN was HNF material which did not absorb to a diethyl- aminoethyl-agarose (DEAE-BioGel A, Bio-Rad Laborato-

ries, Richmond, CA) column equilibrated with 0.1% Triton X-100 in 0.01 M sodium phosphate buffer, pH 7.2. The F was HNF material which did not absorb to a hydroxylapatite (BioGel HTP, Bio-Rad) column equilibrated with the same buffer. The yield of both F and HN was over 80% from the 2 columns. Triton was exchanged for DC as described above. Purity was determined by SDS-PAGE (data not shown and 171).

2.4 Tumor cells and media used

P815 (H-zd) mastocytoma and EL4 (H-2') lymphoma were maintained by weekly i.p. transfers of 1.0 x lo6 ascites cells in mice of the strains of origin, DBAI2 and C57BLI6, respectively. RPMI1640 was supplemented with 10% fetal calf serum, L-glutamine, penicillin and streptomycin [ 111.

2.5 Production of CTL

Mice, 8-12 weeks old, were inoculated i.p. with SV or liposomes containing purified viral glycoproteins. The immunized animals were killed 6 8 days later, and immunized spleen cells were assayed for CTL lytic activity as described below.

Secondary anti-SV CTL were elicited by using responder spleen cells from mice primed 4-6 weeks earlier with 100 1.18 of UV-inactivated SV (protein determination by the method of Lowry [12]) by an i.p. injection.

Suspensions of SV-primed spleen cells were treated with NH4C1 to remove red blood cells 171 and were used as the responder cells for the in vitro generation of CTL. Three x 106 stimulator tumor cells (P815 or EL4, X-irradiated 10000 rd, Picker) or 3 X lo6 nonirradiated syngeneic spleen cells (red blood cells removed with NH4CI) were prein- cubated as described previously (71 with 30 pg of UV-inactivated SV (SV protein determined by the method of Lowry, [21]) unless otherwise stated. For the elicitation of anti-SV CTL, 7 x lo6 SV-primed responder spleen cells were incubated with 3 x lo5 SV-coated X-irradiated tumor cells (10000 rd) or 3 x 10' SV-coated, nonirradiated syngeneic spleen cells for 5 days in 2.0 ml of supplemented RPMI 1640 medium, in 1.7 X 1.6 cm wells (Linbro Scientific Co., Inc., New Haven, CT) in an atmosphere of 6% C 0 2 and 94% air.

When SV glycoproteins, incorporated into liposomes, were used to elicit a secondary anti-SV CTL response, 4 X lo6 SV- primed syngeneic spleen cells were incubated in 0.5 ml of RPMI 1640 containing liposomes for 1 h at 37°C with con- tinual agitation. After 1 h incubation, the mixture was washed by centrifugation (450 x g, 5 min, 3 X) and diluted to a final volume of 2.0 ml and plated in 1.7 x 1.6 cm wells (Linbro) for 5 days with 8.0 x lo6 SV-primed spleen cells. After 5 days of incubation, the cells were harvested, washed by centrifugation (450 x g), counted and resuspended in supplemented RPMI 1640 at various cell concentrations for the cytotoxicity assay (see Sect. 2.6). Viability of the cells after 5 days of incubation was determined by trypan blue exclusion and was always greater than 30%. All effector cells tested in this study were shown to be T cells by the susceptibility of the cytolytic activity of these effector cells to anti-@ antiserum in the presence of complement (C) .

Eur. J . Imrnunol. 1980.10: 923-928 Elicitation of anti-viral cytotoxic T lymphocytes 925

2.6 Cytotoxicity assays

51Cr-labeled target cells (1.0 x lo4) were mixed with serial dilutions of effector spleen cells in a total volume of 200 1.11 of supplemented RPMI 1640. This mixture was incubated 4-6 h at 37°C in an atmosphere of 6% COz and 94% air. The assay was stopped by diluting with cold phosphate-buffered saline (PBS) and the amounts of radioactivity in the supernatant and pellet determined [ 121. The percentage of specific release (Spec. rel.) was calculated as 100 (E-c/l-c) were E is the fraction of 'lCr released by antigen-stimulated effector cells and c is the fraction of 'lCr released by a mock-stimulated responder population. Percentage of release was calculated as 100 X (E/ T) where E is the amount of radioactivity released by target cells incubated with antigen-stimulated effector cells, and T represents the total amount of radioactivity in each assay tube.

Anti-SV antibody titers were determined by inhibition of hemagglutination of chicken red blood cells by SV, as described by Scheid and Choppin [8].

2.7 Target cells

Tumor cells (5.0 x lo6) were incubated with 200 pCi (=7.4 MBq) of Na2lCr O4 (New England Nuclear, Boston, MA) in 0.5 ml of supplemented RPMI 1640 for 1.5 h at 37 "C in an atmosphere of 6% COz and 94% air [ l l ] . Target cells were made susceptible to lysis by anti-SV CTL by incubating at 4 "C for 60 min with different amounts of UV-inactivated SV in 1.0 ml RPMI 1640 [7, 91. The standard SV target cells were 10 pg of UV-inactivated SV grown in embryonated eggs incubated as described previously [7, 91 with 3 x lo6 cells, unless otherwise stated (P815-SV, EL4-SV).

2.8 Reconstitution of viral antigens into liposomes

In a typical preparation, 200 pg of egg lecithin plus cholesterol (30% wtiwt, Calbiochem, San Diego, CA) dissolved in chloroform was dried in the form of a film under Nz. This film was then dissolved into a solution containing 0.2% DC SV glycoproteins at a lipid-to-protein ratio of 1 : 1 in a final volume of 4 ml. Protein determinations were done as described by Lowry et al. [12]. This solution was then dialyzed for 3 W 8 h against PBS at 4 "C or at room temperature for 24-36 h. The dialysate was recovered as an opalescent solution with >80% of the protein associated with the vesicles [12-161.

Figure 1 . Electrophoretic characterization of the purity of 'Z51-lac- toperoxidased, labeled viral glycoproteins as analyzed by SDS-PAGE and identified by autoradiography. HNF (lane A), HNFo (lane B), HN (lane C ) , F (lane D).

3 Results

3.1 Purity of protein reagents

In Fig. 1, an electrophoretic analysis is shown of the viral proteins reconstituted into phospholipid vesicles. The HNF, HNFo, HN and F glycoproteins were found to be essentially pure. The liposomes into which these glycoproteins are incorporated, are unilamellar, 450-850 A in diameter, and 7@80% of the antigenic sites on the HN and F glycoproteins are exposed to the outside of the vesicles (data not shown and [7, 161).

3.2 Elicitation of primary in vivo anti-SV effector cells and antibodies

Table 1 is a composite of results indicating the ability of intact SV or liposomes containing different viral glycoproteins to elicit anti-SV effector cells in vivo. SV grown in MDBK cells, activated with trypsin and rendered uninfectious with UV light (SV-T), elicited a small anti-SV response (Table 1). SV with Fo (SV-MDBK) was not as effective in elicitation of the anti- SV effector cells and was only capable of eliciting an anti-SV effector response at high immunizing doses. Liposomes containing HNF glycoproteins (> 50 pg of protein [12]) were capable of eliciting anti-SV effector cells. Liposomes containing only HN or F glycoproteins were found not to elicit anti-SV effector cells. HNFo-containing liposomes were not as effective as HNF liposomes in eliciting anti-SV effector cells. Lipo- somes containing HNFo glycoproteins injected at higher doses, however, did result in a small amount of lysis. As shown in Table 2, anti-SV effector cells elicited by HNF-reconstituted liposomes were Thy-1, 2+, Ly-1-, Ly-2'.

All viral and liposome preparations tested were found to elicit anti-SV antibodies, as shown in Table 3. These results suggest that F is required for optimal elicitation of primary anti-SV effector cells, while this requirement is apparently not needed for anti-SV antibody formation.

3.3 Elicitation of secondary in vitro anti-SV CTL

Because the in vivo induced primary cytolytic activity was low, we attempted to analyze the priming capacity of the viral (SV- MDBK and SV-T) and liposome (HN, F, HNF and HNFo) preparations, using secondary cultures. Spleen cells primed with either SV or reconstituted liposomes were incubated with syngeneic spleen cells coated with UV-inactivated SV. After 5 days in culture, the effector cells were harvested and analyzed for anti-SV cytolytic activity (Table 4). Spleen cells from mice primed with SV-T or HNF-reconstituted liposomes and cul- tured with SV-coated stimulator cells had strong cytolytic activity (Table 4). The effector cells were shown to be T cells by their sensitivity to anti-Thy-1.2 in the presence of C (data not shown). Spleen cells primed with SV-MDBK, HN, F, HNFo, or HN and F (reconstituted into separate 1iposomes)- reconstituted liposomes were not as effective in priming for elicitation of secondary anti-SV CTL. In fact, the complete lack of a functional fusion complex (F, HN or HN and F liposomes) did not result in any detectable priming above that seen for normal spleen cells.

926 M. McCee, A. H. Hale and M. Panetti Eur. J . Immunol. 1980.10: 923-928

Table 1. In vivo primary elicitation of anti-SV effector cellsa)

Mouse strain Antigen Tor Protcin % Spccific "Cr relcaw ( E : immunization (ug) PSIS PsIS-SV E1-4

HAl.B/c

HALB . B

Nonc sv-1'

SV-MDBK HNF liposonic

HNFo lipowme

HN + F liposomc HN liposonie F liposomc

None sv-7'

SV-MDBK HNF liposomc

HNFo lipowme

HN + F liposomc HN lipowme F liposornc

(BALBlc X HALB. B)F, Nonc SV-T

SV-MDBK HNF liposonie

HN + F liposonie HNFo liposomc

Table 2. Ly phenotype of anti-SV CTL")

Treatment

None Normal mouse serum Anti-Thy-1.2 Anti-Ly-l An ti-Ly-2 Normal mouse serum Anti-Thy-1.2 + C Anti-Ly-l + C Anti-Ly-2 + C

+ C

0.7 0 . 3 I .2 10 1.2 - 13.3 1.7

200 1.1 6.7 0. I IOU 0.8 - 21 .x 1.2

10 0.7 ~ 10.8 1.1 -

IOU 1.1 1S.6 - 1 . 1 1 ?o() I .2 18.7 I . 3

10 1 .o 1 . 1 I .2 lot) 0.8 0. 1 1.3 200 1 . 1 s . 7 1 .4 I(H) -1 .0 0.7 0 . 3 100 0.4 0.S I .2 3 M ) 0.8 1.7 2 .1

-0.7 1.2 1.3 10 0.9 1 .9 0.0

I (HJ 1.8 - ( l . l 0 . X

100 0.5 0.9 -0 .6 I0 1.1 0.6 I .3

I00 1 . 1 1.7 I .7 2(H) -1.7 0.8 0.7

I0 0 . 8 0.9 0.8 100 -0.6 0.7 1.2 200 1 .4 1 . 1 - 1 . 1 2IX) I .o 0.7 -0 .7 200 I .3 1.1 0.0 200 1 .s 0.7 0.4

1 .o 1 . 1 -0.7 I ocl 1.7 21.2 - 0 . 3 2llo I . S n.cl (1.4 IOU 0 .3 S.2 0.7 200 -1).9 10.7 -1.2 2lH) 0.8 I .2 1.7 lo() 1.2 2.1 0.4 ?(MI 1.3 g -0 .6

-

'7 Spec. "Cr re1 ( E : T = I ( l 0 : 1) of PSIS-SV"'

21,s 20.7 19.6 18.7 19.4 16.4

I .4 19.8

1 .9

T=50: I ) EL 4-sv

I .o 1.2 0.7 0 . 3 I .8 0.0 I . o 0 . 8 I .7 1 2 . 0 . 2 0 4

- 0,s

- 1 . 1 11.7 32. I 13. I 11.7 17.1 3.3.7

7.1 (.z 9.7 - 0.4 0 . 3 I .2 (1.4

'1.7

(.7 5.2

15.x 0.4 0.7 3 . 0

-

-

a) BALBlc, BALB . B or (BALBlc X

BALB . B)F, mice were immunized i.p. with serial dilutions of different UV-inactivated viral and subviral fractions. HN + F liposome indicates that the HN + F glycoproteins were incorporated into separate liposomes and injected into a mouse at a dose of 100 pg each. SV-T refers to MDBK-grown SV inactivated with UV-light and incubated with trypsin. SV-MDBK refers to MDBK-grown SV not treated with trypsin. Six to 7 days later, spleen cells were harvested and assayed on P815 or EL4 tumor cells, or on P815 and EL4 tumor cells modified with UV-inactivated SV (P815-SV or EL4-SV). The % release of P815, P815-SV, EL4, or EL4-SV was 6.7, 11.7, 14.7 and 15.7%, respectively. Each value represents 6 measurements with standard deviations (SD) never ex- ceeding 1.7%.

a) BALBic mice were immunized with HNF liposomes (100 pg of total protein) by an i.p. injection. Six days later, the spleen cells were harvested and tested for ability to lyse P815 cells modified with UV-inactivated SV. Before testing for the presence of cytolytic activity, 1.0 X 10' spleen cells were incubated with a predeter- mined optimal dilution of anti-Thy-1.2, anti-Ly-1 or anti-Ly-2 in the presence or absence of rabbit C; (all antisera and C were from Cederlane Laboratories, Ontario, Canada). The cells were washed by centrifugation (SO0 x g) and tested for cytolytic activity. P815- SV target cells had a spontaneous 5'Cr release of 17.9%.

b) Each number represents the average of 6 determinations with SD deviation for all measurements of not greater than 3.2%.

Eur. J. Immunol. 1980.10: 923-928

Table 3. In vivo primary elicitation of anti-SV antibodies

Mouse strain Antigen for"' immunization

BALBlc

BALB . B

None sv

HNF liposomes

HNFo lipoaornes

H N liposornca F liposomes

None sv

HNF liposornes

HNFo liposornes

HN liposonies F liposornes

None sv

HNF liposonic

HNFo liposome

(BALBlc x BALB . B)Fl

Inhibition of hernag- glutination

titer'"

< 30 XO

320 < 20

100 320

< 20 1 ho 320 320 80

< 20 80

I60 40

160 320 10

160 320 320 I60

< 20 320 I60 320

80 320

a) Mice were immunized i.p. with different amounts of UV-inactivated SV or subviral liposome preparations and tested 10 days later.

b) The reciprocal of the final dilution of antiserum to give inhibition of agglutination. Twofold dilutions of antisera were done.

3.4 Generation of primary in vitro anti-SV CTL response

As indicated in Table 4, a small stimulation of anti-SV CTL activity is always noted for both normal as well as spieen ceils primed with HN or F-containing liposomes. HN or F-containing liposomes did not appear to prime the spleen cells described in Table 4, since no increase in cytolytic activity could be found that was higher than that observed with normal spleen cells incubated with the SV-coated spleen cells. There- fore, this activity apparently represents a primary in vitro response to the SV-coated spleen cells. The elicitation of primary in vitro anti-SV CTL was recently reported by Kos- zinowski et al. [5] and Jung et al. [HI. Because a requirement for an active fusion complex was indicated in our previous experiments (Tables 1 and 4 ) , we tested these same viral and subviral fractions for their ability to elicit an in vitro primary anti-SV CTL response (Table 5). These results are essentially the same as those found for the in vivo response, i.e. an active fusion complex (HNF) is more effective in eliciting an anti-SV CTL response (Table 5). Some differences between the in vivo and in vitro results were observed. HN-containing liposomes

Elicitation of anti-viral cytotoxic T lymphocytes Y21

Table 4. Elicitation of secondary anti-SV CTL")

Antigen f o r Protein c+ Spec. "Cr re1 "' priming (I%) (E : 7' = 50 : 1)

PXIS P 815-sv

None SV-T

SV-MDBK

HNF liposome

HNFo liposome

HN + F liposorne HN liposorne F liposome

0 . Y 1 . 1 0.2 0.6 1.4 I .7 1.1 0.4 0.7

-0.4 0 . 8

-1.1 0.7 0 .4

11.3 3s.x 78.7 12.7 22. I 3s.x 32.7 03. I 12.1 IX.8 25.8 12.3 lo.x 1O.Y

a) BALBic mice were immunized i.p. with viral and subviral fractions a t the levels indicated. Four to 8 weeks later, their spleen cells were harvested and incubated with SV-coated BALB/c spleen cells at the optimal responder to stimulator ratio (2 : 1) as described in Sect. 2.5. Five days later, the cells were harvested and tested on P 815 or SV-modified P 815 target cells.

b) The spontaneous 'lCr release of P815 and PUS-SV was 11.7 and 15.6%, respectively.

Table 5. Generation of primary anti-SV CTL"'

Antigen ProteIni10' cells ci spec. "Cr re1.l" (I%) ( E : T = 5 0 : I )

P x15-sv PX15

SV-T 1 .O 10.0

100.0 SV-MDBK 1 .o

10.0 1 o(J. 0

HNF I .0 10.0

1 00.0 HNFo 1 .o

10.0 100.0

HN 10.0 1 00.0

F 10.0 l(M.0

(50.0 + 50.0) HN + F ( ? . O + 5.0)

1.1 0.8 2.2 0.6 1.2

-1.0 0 . Y

-1.3 1 .o 1.4

-1.7 -0.Y -0.X

1.2 0.2 1 . 1 0.1 1.4

I .z 15.9 - 42.7

1.3 0.2 16..1

0.7 13.8 33.7

0 . I 2.1

17.1 -0.8 10.7

1.3 2.6 1 .z - 7.9

a) Normal spleen cells were incubated with stimulator cells modified as described in Sect. 2.5 with different amounts of viral and subviral fracti0nsll0~ cells at the optimal responder to stimulator ratio (2: 1). After 5 days in culture, the spleen cells were harvested and assayed on P 815 or SV-modified P 815 target cells.

b) The spontaneous "Cr release for P815 and PUS-SV was 15.7 and 18.9% respectively.

928 M. McGee, A . H. Hale and M. Panetti Eur. J. Immunol. 1980.10: 923-928

were able to elicit a low primary response of high concentrations. However, F-containing liposomes did not elicit a detectable anti-SV response.

4 Discussion

When viruses infect host cells, they may synthesize viral proteins which become integrated into the host cell’s plasma membranes and are presented to the immune system in the context of normal host cell surface components (i.e. H-2K and/or H-2D gene products). Viruses which have been inactivated by a variety of methods (irradiation, chemical inactiva- tion, etc.) cannot synthesize their viral glycoproteins and thus, presentation of virus-specific antigens to the immune system is restricted, Although virus-specific antigen presentation ap- pears to occur for humoral responses, many inactivated viruses are unable to elicit CTL. In this report, we have reconstituted viral proteins into phospholipid vesicles in order to evaluate the requirement for integration of viral antigens into host cell membranes for elicitation of anti-viral CTL.

Liposomes reconstituted with the major surface glycoproteins of SV (HNF) were effective elicitors of in vivo and in vitro primary anti-SV CTL. However, the incorporation of Fo or its absence in HN-containing phospholipid vesicles reduced the ability of these liposomes to elicit either an in vivo or an in vitro primary anti-SV CTL response. Experiments are in progress to specifically evaluate if indeed the HNF liposomes do fuse with cells in culture. We have no direct evidence of fusion at this time. However, the reduced ability of HN or F glycoproteins incorporated alone into separate liposomes to elicit anti-viral CTL is consistent with the requirement of both glycoproteins for fusion [8]. The ability of HN or HNFo liposomes at high concentrations to elicit an in vitro primary anti- SV response suggests that other mechanisms of antigen presentation may also exist. Experiments are in progress to elabo- rate what those mechanisms might be and what cells might be involved. All liposome preparations were found to elicit anti- SV antibodies after an i n vivo immunization. These results are significant because they suggest that integration of viral antigens into host cell membranes is not an absolute requirement

for elicitation of anti-viral antibodies. Although we have no direct evidence for fusion, our data is suggestive that a requirement for integration of viral antigens into host cell membranes does exist for optimal elicitation of anti-viral CTL. Thus, in summary, these results suggest that the requirements for the presentation of virus-specific antigens to the immune system may differ between CTL and humoral responses.

We are grateful for the excellent secreiarial assistance of Mss. Jean Hooker, Inga Floyd-Kear and Janet Lowry.

Received April 30, 1980; in revised form August 15, 1980.

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3 Palmer, J. C., Lewandowski, L. J. and Waters, D., Nature 1977.

4 Wiktor, T. J., Doherty, P. C. and Koprowski, H., Proc. Natl.

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13 Engelhard, V. H., Strominger, J . L., Mescher, M. and Burakoff,

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Elicitation of primary anti-Sendai virus cytotoxic T lymphocytes with purified viral glycoproteins

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