Immunology 1978 34 763
Primary in vitro sensitization of virus specific cytotoxic T lymphocytes H. JUNG, K. PFIZENMAIER, ANNA STARZINSKI-POWITZ, M. ROLLINGHOFF & H. WAGNER Institut fuir Medizinische Mikrobiologie der Johannes Gutenberg-Universitdt Mainz, Hochhaus am Augustusplatz, D-6500 Mainz/ W. Germany
Received 28 July 1977; acceptedfor publication 18 August 1977
Summary. CBA mouse-derived splenic lymphocytes treated with either fi-priopionlactone-inactivated or u.v. light-inactivated parainfluenza (sendai) virus stimulated in vitro unprimed syngeneic T-lymphocytes to differentiate into cytotoxic T-lymphocytes (CTL). The CTL generated were virus specific and H-2 restricted. For optimal CTL responses to be induced (i) a critical treatment of stimulator cells, (ii) an optimal ratio of responder to stimulator cells and (iii) an in vitro incubation period of 5 days was required. The in vitro system for the induction of primary virus-specific CTL responses may be useful to analyse the sensitization phase of H-2 restricted virus-specific CTL. INTRODUCTION In the course of a murine virus infection cytotoxie thymus-derived (T) lymphocytes (CTL) are generated capable of interacting maximally with virus infected targets provided they share H-2K or H-2D region determinants with the effector cells (Doherty, Zinkernagel & Ramshaw, 1974; Blanden, Pang & Dunlop, 1977; Koszinowski & Ertl, 1975; Pfizenmaier, Trostmann, Rollinghoff & Wagner, 1976a). One explanation for this phenomenon is that the
cytotoxic T-cells express two distinct receptors with specificity for self (H-2) and non-self (virus) antigens (Doherty et al., 1974; Doherty, Gotze, Trinchieri & Zinkernagel, 1976; Janeway, Wigzell & Binz, 1976; Zinkernagel, 1976; Pfizenmaier, Starzinski-Powitz, Rodt, Rollinghoff & Wagner, 1976b). An alternative is that the T-cell receptors are specific for an 'altered self' antigen created by the interaction of virus and H-2 molecules (Doherty et al., 1976; Zinkernagel, 1976). Although the evidence for either of both concepts is still equivocal, it is generally agreed that the specificity of the virus immune CTL is dictated during the sensitization phase of the antigen-reactive T-cells (Doherty et al., 1974; Zinkernagel, 1976; Pfizenmaier et al., 1976b). This phase however is difficult to analyse in vivo; our aim therefore was to establish an experimental system which allows us to analyse the induction of virus-specific CTL under defined in vitro conditions. Probably due to the cytopathic effect of infectious viruses on in vitro cultured cells, our initial attempts to induce in culture virus-specific CTL were unsuccessful. Using the parainfluenza (sendai) virus system we (Pfizenmaier, Starzinski-Powitz, Wagner & Rollinghoff, 1977a) as others (Schrader & Edelman, 1977; Koszinowski, Gething & Waterfield, 1977) noted that sendai virus-specific CTL do lyse syngeneic targets passively coated with inactivated, non-infectious sendai virion material. We thus asked whether cells passively coated with non-
Correspondence: Dr H. Jung, Institut fur Medizinische Mikrobiologie der Johannes Gutenburg-Universitat Mainz, Hochhaus am Augustusplatz, D-6500 Mainz, W. Germany. 0019-2805/78/0400-0763 502.00 (©1978 Blackwell Scientific Publications
763
H. Jung et al.
764
infectious sendai viruses would trigger in vitro sendai virus-specific CTL.
MATERIALS AND METHODS Mice CBA and BALB/c mice were purchased from G.I. Bomholtgaard, Ry, Denmark and used for the experiments within 10 days after arrival. The B10, B1O.A, B10.(4R) and B10.D2 mice used were from our own breeding facilities.
Culture system The methods of preparing a single cell suspension, the culture medium and the culture method used have been described in detail (Wagner, Gotze, Ptschelinzew & Rollinghoff, 1975). In brief 4 x 106 splenic responder cells and 0-5-1 x 106 stimulator cells were cultured in a volume of 2 ml culture medium (DMEM, H-16, Gibco, Grand Island, N.Y.) supplemented with 5 % foetal calf serum and 5 x 10-5 M 2-mercaptoethanol in multi dish culture trays (FB-24Tc, Linbro Chemicals, New Haven, Conn., U.S.A.).
Inactivated sendai virus Two batches were used: (i) infectious sendai virus (107 EID3o) in 5 ml gelatine saline was exposed to 40 W germicidal u.v. light inactivation for 45 min at room temperature at a distance of 10 cm. Thereafter no remaining infectious virus was detected in a plaque assay using secondary chicken fibroblasts (kindly performed by Dr Punter, Farbwerke Hoechst, Frankfurt/W. Germany). The inactivated sendai virus suspension had 103 HAU/ml. (ii) In most of the experiments a commercially available fi-propionlactone-inactivated sendai virus suspension known as 'fusion factor' (Lot. No. 138-1, Connaught Laboratories, Ltd., Toronto, Canada) was used. This virus suspension had 10' HAU per ml.
Preparation of target cells Primary peritoneal macrophages (Pfizenmaier et al., 1976a) were incubated for 1-4 h with either infectious (lOEIDso per cell) or for 1 h with inactivated sendai virus suspension (103 HAU), two times washed and labelled with 51Cr-chromate (Amersham-Buchler, Frankfurt, W. Germany) as described (Pfizenmaier et al., 1976a).
51Cr-cytotoxicity assay The cultured cells were harvested, and their viability was determined using the eosin dye exclusion method. A graded number of viable 'attacker cells' were mixed with a constant number (2 x 104) of 51Cr-labelled macrophages in 1 ml DMEM in round bottom plastic tubes (Fa. Greiner, Nuirtingen, W. Germany, Cat. No. 107), centrifuged for 1 min at 300g and processed as described (Wagner & Rollinghoff, 1974). The percent specific lysis was calculated according to the formula 5ICr-release by 5'Cr-release by specific immune cells normal cells 100 ~~~~~~~~x o/ = 51Cr-release by maximal ' cells normal lysis 51Cr-release Standard deviation (s.d.) of the experimental samples were calculated. The data are presented without s.d. since in the experiments listed, the s.d. of % specific lysis was less than 5 %.
Complement-dependent cytotoxic test for Thy 1.2 positive lymphocytes The AKR anti-CBA thymocyte serum used had a cytotoxic titre of 1: 32 when assayed towards peripheral T cells. The cells to be tested were suspended in 0 5 ml of anti-Thy 1.2 serum (dilution 1: 4) and incubated for 30 min at 37°. The cells were washed and resuspended in 1 ml of agaroseabsorbed guinea-pig serum (final dilution 1: 6). After incubation for an additional 30 min at 370, the cells were washed, and assayed for cell-mediated
cytotoxicity. Preparation ofstimulator cells 2 x 107 spleen cells depleted of erythrocytes as described elsewhere (Doherty & Zinkernagel, 1976) were incubated with a suspension of inactivated sendai virus containing 102_103 HAU in a total volume of 2 ml for 1 h at 37°. Thereafter the cells were X-irradiated with 2000 rad (Philips machine RT 200) and washed twice.
Measurement of delayed type hypersensitivity (DTH) to sendai virus Using the method of Tosolini & Mims (1971) mice were selected from the groups used experimentally and tested for DTH reactivity towards sendai viruses. In none of the CBA mice tested was a positive reactivity observed.
Virus specific CTL induced in vitro
765
Table 1. In vitro induction of sendai virus immune cytotoxic effector cells % Specific lysis of CBA macrophagest
Untreated Responder cells CBA CBA CBA
Stimulator* cells None CBA infected CBA treated with non-infectious virus§
Infected
Externally coated with inactivated virus§
t50:1
5:1
50: 1
5:1
50: 1
2 0
1 2
4 19
-1 11
6
6
2
61
47
50
5: 1 2 N.D. 24
* Ratio responder cells and stimulator cells was 6: 1. t Ratio effector cells to target cells. $ Background lysis of the target cells during the 3 h cytotoxicity assay was less than 34%4 § I-propionlactone-inactivated sendai virus.
RESULTS
Membrane-associated infectious sendai virus antigens induce virus specific CTL in vitro. There is evidence that sendai virus-specific CTL not only lyse sendai virus-infected syngeneic target cells but also syngeneic cells coated with non-infectious sendai virus particles (Schrader & Edelman, 1977; Koszinowski et al., 1977; Pfizenmaier et al., 1977a). Since these data suggested that inactivated virus material is sufficient to modify a cell and make it a specific 'target' for sendai virus immune CTL, we tested the possibility of using syngeneic cells coated with inactivated sendai virus in order to initiate in vitro primary anti-sendai virus CTL responses. For this purpose in a first step CBA mouse splenic lymphocytes were incubated in vitro with either infectious, or inactivated sendai virus for 60 min at 370 and washed (stimulator cells). In a second step CBA mouse splenic lymphocytes (4 x 106 responder cells) were co-cultured with 1 x 106 of stimulator cells. After 5 days the cultured cells were assayed for cytotoxicity towards sendai virus infected syngeneic macrophages. Representative results obtained are given in Table 1. Accordingly strong cytotoxicity could be generated using stimulator cells coated with inactivated sendai virus, whereas cultures containing sendai virus-infected stimulator cells generated only weak cytotoxicity. The data in Table 1 also indicate that sendai virus immune CTL generated in vitro lysed fl-propion lactone sendai virus treated target cells to a similar extent as target cells incubated with live virus. Thus both the afferent
limb and the efferent limb of a primary cytotoxic anti-sendai CTL response could be basically induced in vitro using inactivated sendai virus material.
Specificity of the cytotoxic activity generated Next, various parameters of the primary in vitro anti-sendai virus CTL response were defined. From the dose-response curve (Fig. 1) of stimulator cell formation using dilutions of 8-propionlactoneinactivated sendai virus, it can be deduced that about 100-1000 HAU equivalents of inactivated sendai virus material per 107 lymphocytes appeared to be optimal. Using stimulator cells obtained by treating CBA spleen cells (107) with 1000 HAU equivalents of inactivated sendai virus and titrating the optimal ratio of responder to stimulator cells for generating in vitro CTL responses, results as given in Fig. 2 were obtained. Accordingly, a ratio responder to stimulator cells of 4-8 to 1 yielded the highest CTL responses. Moreover, X-irradiating the stimulator cells made no obvious difference (Fig. 2). In Fig. 3 representative results of the kinetics of the induction of anti-sendai immune CTL are given. Similar to primary cytotoxic allograft reactions (Cerottini & Brunner, 1974; Wagner, Rollinghoff & Nossal, 1973), peak cytotoxic activity generated was observed at day 5 of culture. That the cytotoxic activity generated in vitro was due to T lymphocytes was strongly suggested by the observation that treatment of the effector cells with anti-Thy 1.2 serum plus complement abrogated their cytotoxic activity
(Table 2).
H. Jung et a!.
766 90
70(
70~~~ 70
aJ 4)
-30 3
*
i/
3 ___,_A_
10
.^A
AJI!
0
A
100
10
HA- titre
104
I0
ner 107 stimulotor
cells
Figure 1. Immunogenicity of syngeneic lymphocytes treated with a graded concentration of fl-propionlactone-inactivated sendai virus material. Splenic lymphocytes of CBA mice were divided into two parts. Aliquots (10' cells) of one part was incubated (30 min at 370) with a graded concentration of inactivated sendai virus (stimulator cells). The untreated cells (responder cells) were cultured together with the washed stimulator cells for 5 days. The cultured cells of the various groups were harvested and assayed for cytotoxicity in a 3 h 51Cr assay, towards CBA macrophages (A); sendai virus-infected CBA macrophages (0); and CBA macrophages treated with inactivated sendai virus (U) at an effector to target cell ratio of 50: 1. Background lysis of the targets was less than 31%.
80 r- (a) I
--
60 ~n -'
4
4 5 6 7 Days of culture
8
10
9
Figure 3. Kinetics of the generation of cytotoxicity. CBA splenic responder cells were cultured with A-propionlactoneinactivated sendai virus treated (103 HAU per 107 cells) CBA spleen cells (stimulator cells). At daily intervals groups of the cultures were harvested and assayed for cytotoxicity towards sendai virus-infected (0) or normal (A) CBA macrophages at an attacker to target cell ratio of 50: 1. Background lysis of the target cells during the 3 h assay was less than 31 %.
Table 2. Effect of anti-Thy 1.2 serum plus complement upon in vitro immunized cytotoxic lymphocytes
% Specific lysis of sendai virus-infected CBA-macrophages* Treatment of effector cells
50: 1
5: 1
None Anti-Thy 1.2 G.P. complement Anti-Thy 1.2 plus complement
78 77 70 9
49 42 25 7
* Ratio effector to target cells; assay time 3 h; background lysis 13 %.
40
20
1:1
4:1 8:1 40:1 1:1 4:1 8:1 Ratio resoonder to stimulator cells
40:1
Figure 2. Dose-response relation of cytotoxic activity generated in vitro: CBA splenic responder cells (4 x 106) were cultured with various numbers of stimulator cells (CBA spleen cells treated with 103 HAU inactivated sendai virus per 107 cells). At day 5 of culture the stimulated CBA cells were assayed in a 5'Cr-release test (3 h) towards sendai virus infected CBA macrophages. Background lysis of the target cells was less than 22%. (0) Ratio effector cells to target cells of 50: 1. (0) Ratio of 5: 1. (a) Non-irradiated stimulator cells; (b) X-irradiated (2500 rad) stimulator cells.
Parameters of primary in vitro anti-sendai virus CTL responses To test for the virus specificity of the CTL generated in vitro sensitized sendai immune CTL were tested for lytic activity towards syngeneic targets infected with either sendai virus, LCM virus or Herpes Type 1 virus (Pfizenmaier, Starzinski-Powitz, Rollinghoff, Falke & Wagner, 1977b). Similar to in vivo generated sendai immune CTL, in vitro induced sendai immune CTL exhibited a strict specificity for syngeneic targets infected with the virus used for the induction of the immune response (Table 3). In addition, in vitro generated sendai virus immune CTL did not
Virus specific CTL induced in vitro Table 3. Specificity of in vitro induced antisendai virus immune CTL
did not show any detectable DTH reactions to inactivated sendai virus (data not shown). To bypass virus-induced cytopathic effects within the cultures, we took advantage of the observation that non-infectious inactivated sendai virus particles are able to modify syngeneic cells and make them specific targets for sendai immune CTL (Pfizenmaier et al., 1977a; Schrader & Edelman, 1977; Koszinowski et al., 1977). Consequently we argued that syngeneic lymphocytes, the cell membrane of which had been fused with inactivated sendai virus particles may trigger in vitro primary sendai virus specific CTL responses. Indeed using both u.v. light irradiated or f,-propionlactone-inactivated sendai virus material at a concentration of about 100-1000 HAU per ml to treat 107 CBA splenic lymphocytes (stimulator cells), strong cytolytic activity towards both sendai virus infected and p8-propionlactoneinactivated sendai virus treated target cells was generated in vitro. Moreover, there was a clear doseresponse relationship between the concentration of inactivated sendai virus used for the treatment of stimulator cells and their immunogenicity (Fig. 1). In addition a critical ratio of responder to stimulator cells was required in order to induce efficient sendai virus-specific CTL (Fig. 2). Finally the CTL generated in vitro exhibited the same features as in vivo sensitized CTL, that is they were virus specific and H-2 restricted. It is concluded therefore that the in vitro approach described results in the induction of virus specific CTL functionally similar to those activated under in vivo conditions. Thus the system may be of advantage in dissecting and analysing the sensitization phase of CTL precursor cells reactive with specific viral antigens on H-2 restricted target
% Specific lysis of CBA macrophages Virus strain used for infection of targets None LCM Herpes Typ 1 Sendai
50: 1*
5: 1
4 3 -2 70
2 0 1 29
767
* Ratio of in vitro sensitized CBA anti-sendai virus immune CTL (attacker cells) to target cells. Background lysis of the target cells was less than 32 %.
lyse allogeneic target cells infected with sendai virus (Table 4) thus demonstrating the same H-2 restriction as in vivo induced virus immune CTL (Doherty & Zinkernagel, 1976; Pfizenmaier et al., 1977a; Ertl & Koszinowski, 1976). DISCUSSION The aim of the experiments reported was to induce in vitro a primary T-cell-mediated cytotoxic immune response to sendai virus infected cells. To exclude the possibility that the mice used in the experiments had been already in contact with sendai virus and that therefore we would be dealing with an in vitro sensitization of in vivo primed T lymphocytes, the CBA mice used experimentally were killed within 10 days of removing them from SPF conditions. Such mice
Table 4. H-2 restriction of in vitro sensitized sendai virus specific CTL
% Specific lysis of sendai virus-infected macrophages CBA
Sendai virus specificCTL* CBA
B1O(4R) B1O.D2
BALB/c
BlO
BlO.A
B1O(4R)
t50: 1 5:1 50:1 5:1 50:1 5: 1 50:1 5:1 50:1 5:1 48 19 5
12 5 2
9 0 77
6 1 18
1 0 0 -2 N.D.
32 13 34
15 6 3
33 6 13 2 N.D.
* In vitro sensitization was performed as under conditions described in Table 1. t Ratio of attacker to target cells used in the 3 h cytotoxicity assay. Each effector cell population was tested in an independent assay against the target cells listed. Background lysis of the target cells was less than 26 %.
768
H. Jung et al.
cells. The data indicate that, for the induction of sendai virus-specific CTL, no translation of early viral proteins nor expression of viral proteins on the cell surface from 'within' is required. This is in contrast to results obtained in the vaccinia system (Ada, Jackson, Blanden, Tha Hla & Bowern, 1976). Most likely the capacity of inactivated sendai virus to bypass these requirements is due to its unique capacity to mediate cell-cell fusion, which in turn allows by mere incubation with inactivated sendai viruses the insertion of large amounts of virus material into the membrane of both stimulators and target cells. If so, one may deduce that virus-specific CTL can be generated in vitro against a variety of antigens, provided they can be inserted properly into the cell membrane of the stimulator cells and target cells. Various laboratories have succeeded inducing in vitro secondary sendai virus-specific CTL responses (Schrader & Edelman, 1977; Koszinowski et al., 1977; Zweerink, Courtneidge, Skehel, Crumpton & Askonas, 1977). In one report (Schrader & Edelman, 1977) small but significant primary sendai virus specific CTL responses have been described. By analysing the various parameters required for the in vitro induction of virus-specific CTL, we now report cytolytic activity comparable to those obtained by an in vivo immunization regime (Doherty & Zinkernagel, 1976; Ertl & Koszinowski, 1976). Of special interest is the simplicity by which virus-specific CTL can be induced and assayed in a short termed 3 h 51Cr-test. We therefore hope that the in vitro approach described may be useful in analysing the cellular events resulting in vitro in H-2 restricted primary induced virus-specific CTL.
ACKNOWLEDGMENTS The skilled technical assistance of Mrs A. Neumann is gratefully acknowledged. This work was supported by the SFB 107.
REFERENCES ADA G.L., JACKSON D.C., BLANDEN R.V., THA HLA R. & BOWEN N.A. (1976) Changes in the surface of virusinfected cells recognized by cytotoxic T cells. I. Minimal
requirements for lysis of ectromelia-infected P-815 cells. Scand. J. Immunol. 5, 23. BLANDEN R.V., PANG T.E. & DUNLOP M.B.C. (1977) T cell recognition of virus-infected cells. In: Cell surface reviews,
vol. 2 (Ed. by G. Poste and G. L. Nicolson), p. 249. North Holland, Amsterdam. CERorrINI J.C. & BRUNNER K.T. (1974) Cell-mediated cytotoxicity, allograft rejection, and tumor immunity. Adv. Immunol. 18, 67. DOHERTY P.C., ZINKERNAGEL R.M. & RAMSHAW I.A. (1974) Specificity and development of cytotoxic thymus-derived lymphocytes in lymphocytic choriomeningitis. J. Immunol. 112, 1548. DOHERTY P.C., GOTZE D., TRINCHIERI G. & ZINKERNAGEL R.M. (1976) Models for recognition of virally modified cells by immune thymus-derived lymphocytes. Immunogenetics, 3, 517. DOHERTY P.C. & ZINKERNAGEL R.M. (1976) Specific immune lysis of paramyxovirus-infected cells by H-2 compatible thymus-derived lymphocytes. Immunology, 31, 27. ERTL H. & KosZINOWSKI U. (1976) Cell-mediated cytotoxicity against sendai-virus-infected cells. Z. ImmunForsch. 152, 128. JANEWAY C.A. Jr, WIGZELL H. & BINz H. (1976) Two different VH gene products make up the T-cell receptors. Scand. J. Immunol. 5, 993. KosZINOWSKI U. & ERTL H. (1975) Lysis mediated by T cells and restricted by H-2 antigen of target cells infected with vaccinia virus. Nature (Lond.), 255, 552. KOsZINOWSKI U., GETHING M.J. & WATERFIELD M. (1977) T-cell cytotoxicity in the absence of viral protein synthesis in target cells. Nature (Lond.), 267, 160. PFIZENMAIER K., TROSTMANN H., ROLLINGHOFF M. & WAGNER H. (1976a) Cell-mediated immunity in lymphocytic choriomeningitis. I. The specificity of the cytotoxic T lymphocytes. Z. ImmunForsch. 151, 224. PFIZENMAIER K., STARZINSKI-POWITz A., RODT H., ROLLINGHOFF M. & WAGNER H. (1976b) Virus and trinitrophenyl hapten-specific T-cell-mediated cytotoxicity against H-2 incompatible target cells. J. exp. Med. 143, 999. PFIZENMAIER K., STARZINSKI-POWITz A., WAGNER H. & ROLLINGHOFF M. (1977a) T-cell-mediated antiviral cytotoxicity: Cell membrane associated virion antigen is sufficient for lysis of H-2 matched target cells. Z. ImmunForsch. 153, 268. PFIZEMAIER K., STARZINSKI-POWITZ A., ROLLINGHOFF M., FALKE D. & WAGNER H. (1977b) T-cell-mediated cytotoxicity against herpes simplex virus-infected target cells. Nature (Lond.), 265, 630. SCHRADER J.W. & EDELMAN G.M. (1977) Joint recognition by cytotoxic T cells of inactivated sendai virus and products of the major histocompatibility complex. J. exp. Med. 145, 523. TOSOLINI F.A. & MiMs C.A. (1971) Effect of murine strain and viral strain on the pathogenesis of lymphocytic choriomeningitis infection and a study of footpad responses. J. Infect. Dis. 123, 134. WAGNER H., GOTZE D., PTSCHELINZEW L. & ROLLINGHOFF M. (1975) Induction of cytotoxic T lymphocytes against I-region-coded determinants: in vitro evidence for a third histocompatibility locus in the mouse. J. exp. Med. 142, 1477. WAGNER H., ROLLINGHOFF M. & NOSSAL G.J.V. (1973) T-cell-mediated immune responses induced in vitro: A probe for allograft and tumor immunity. Transplant. Rev. 17, 3.
Virus specific CTL induced in vitro WAGNER H. & ROLLINGHOFF M. (1974) T-cell-mediated cytotoxicity: discrimination between antigen recognition, lethal hit and cytolysis phase. Eur. J. Immunol. 4, 475. ZINKERNAGEL R.M. (1976) H-2 restriction of virus-specific cytotoxicity across the H-2 barrier. Separate effector T cell specificities are associated with self-H-2 and with the
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tolerated allogeneic H-2 in chimeras. J. exp. Med. 144, 933. ZWEERINK H.J., COURTNEIDGE S.A., SKEHEL J.J., CRUMPTON M.J. & ASKONAS B.A. (1977) Cytotoxic T cells kill influenza virus infected cells but do not distinguish between serologically distinct type A viruses. Nature (Lond.), 267, 354.
Primary in vitro sensitization of virus specific cytotoxic T lymphocytes H. JUNG, K. PFIZENMAIER, ANNA STARZINSKI-POWITZ, M. ROLLINGHOFF & H. WAGNER Institut fuir Medizinische Mikrobiologie der Johannes Gutenberg-Universitdt Mainz, Hochhaus am Augustusplatz, D-6500 Mainz/ W. Germany
Received 28 July 1977; acceptedfor publication 18 August 1977
Summary. CBA mouse-derived splenic lymphocytes treated with either fi-priopionlactone-inactivated or u.v. light-inactivated parainfluenza (sendai) virus stimulated in vitro unprimed syngeneic T-lymphocytes to differentiate into cytotoxic T-lymphocytes (CTL). The CTL generated were virus specific and H-2 restricted. For optimal CTL responses to be induced (i) a critical treatment of stimulator cells, (ii) an optimal ratio of responder to stimulator cells and (iii) an in vitro incubation period of 5 days was required. The in vitro system for the induction of primary virus-specific CTL responses may be useful to analyse the sensitization phase of H-2 restricted virus-specific CTL. INTRODUCTION In the course of a murine virus infection cytotoxie thymus-derived (T) lymphocytes (CTL) are generated capable of interacting maximally with virus infected targets provided they share H-2K or H-2D region determinants with the effector cells (Doherty, Zinkernagel & Ramshaw, 1974; Blanden, Pang & Dunlop, 1977; Koszinowski & Ertl, 1975; Pfizenmaier, Trostmann, Rollinghoff & Wagner, 1976a). One explanation for this phenomenon is that the
cytotoxic T-cells express two distinct receptors with specificity for self (H-2) and non-self (virus) antigens (Doherty et al., 1974; Doherty, Gotze, Trinchieri & Zinkernagel, 1976; Janeway, Wigzell & Binz, 1976; Zinkernagel, 1976; Pfizenmaier, Starzinski-Powitz, Rodt, Rollinghoff & Wagner, 1976b). An alternative is that the T-cell receptors are specific for an 'altered self' antigen created by the interaction of virus and H-2 molecules (Doherty et al., 1976; Zinkernagel, 1976). Although the evidence for either of both concepts is still equivocal, it is generally agreed that the specificity of the virus immune CTL is dictated during the sensitization phase of the antigen-reactive T-cells (Doherty et al., 1974; Zinkernagel, 1976; Pfizenmaier et al., 1976b). This phase however is difficult to analyse in vivo; our aim therefore was to establish an experimental system which allows us to analyse the induction of virus-specific CTL under defined in vitro conditions. Probably due to the cytopathic effect of infectious viruses on in vitro cultured cells, our initial attempts to induce in culture virus-specific CTL were unsuccessful. Using the parainfluenza (sendai) virus system we (Pfizenmaier, Starzinski-Powitz, Wagner & Rollinghoff, 1977a) as others (Schrader & Edelman, 1977; Koszinowski, Gething & Waterfield, 1977) noted that sendai virus-specific CTL do lyse syngeneic targets passively coated with inactivated, non-infectious sendai virion material. We thus asked whether cells passively coated with non-
Correspondence: Dr H. Jung, Institut fur Medizinische Mikrobiologie der Johannes Gutenburg-Universitat Mainz, Hochhaus am Augustusplatz, D-6500 Mainz, W. Germany. 0019-2805/78/0400-0763 502.00 (©1978 Blackwell Scientific Publications
763
H. Jung et al.
764
infectious sendai viruses would trigger in vitro sendai virus-specific CTL.
MATERIALS AND METHODS Mice CBA and BALB/c mice were purchased from G.I. Bomholtgaard, Ry, Denmark and used for the experiments within 10 days after arrival. The B10, B1O.A, B10.(4R) and B10.D2 mice used were from our own breeding facilities.
Culture system The methods of preparing a single cell suspension, the culture medium and the culture method used have been described in detail (Wagner, Gotze, Ptschelinzew & Rollinghoff, 1975). In brief 4 x 106 splenic responder cells and 0-5-1 x 106 stimulator cells were cultured in a volume of 2 ml culture medium (DMEM, H-16, Gibco, Grand Island, N.Y.) supplemented with 5 % foetal calf serum and 5 x 10-5 M 2-mercaptoethanol in multi dish culture trays (FB-24Tc, Linbro Chemicals, New Haven, Conn., U.S.A.).
Inactivated sendai virus Two batches were used: (i) infectious sendai virus (107 EID3o) in 5 ml gelatine saline was exposed to 40 W germicidal u.v. light inactivation for 45 min at room temperature at a distance of 10 cm. Thereafter no remaining infectious virus was detected in a plaque assay using secondary chicken fibroblasts (kindly performed by Dr Punter, Farbwerke Hoechst, Frankfurt/W. Germany). The inactivated sendai virus suspension had 103 HAU/ml. (ii) In most of the experiments a commercially available fi-propionlactone-inactivated sendai virus suspension known as 'fusion factor' (Lot. No. 138-1, Connaught Laboratories, Ltd., Toronto, Canada) was used. This virus suspension had 10' HAU per ml.
Preparation of target cells Primary peritoneal macrophages (Pfizenmaier et al., 1976a) were incubated for 1-4 h with either infectious (lOEIDso per cell) or for 1 h with inactivated sendai virus suspension (103 HAU), two times washed and labelled with 51Cr-chromate (Amersham-Buchler, Frankfurt, W. Germany) as described (Pfizenmaier et al., 1976a).
51Cr-cytotoxicity assay The cultured cells were harvested, and their viability was determined using the eosin dye exclusion method. A graded number of viable 'attacker cells' were mixed with a constant number (2 x 104) of 51Cr-labelled macrophages in 1 ml DMEM in round bottom plastic tubes (Fa. Greiner, Nuirtingen, W. Germany, Cat. No. 107), centrifuged for 1 min at 300g and processed as described (Wagner & Rollinghoff, 1974). The percent specific lysis was calculated according to the formula 5ICr-release by 5'Cr-release by specific immune cells normal cells 100 ~~~~~~~~x o/ = 51Cr-release by maximal ' cells normal lysis 51Cr-release Standard deviation (s.d.) of the experimental samples were calculated. The data are presented without s.d. since in the experiments listed, the s.d. of % specific lysis was less than 5 %.
Complement-dependent cytotoxic test for Thy 1.2 positive lymphocytes The AKR anti-CBA thymocyte serum used had a cytotoxic titre of 1: 32 when assayed towards peripheral T cells. The cells to be tested were suspended in 0 5 ml of anti-Thy 1.2 serum (dilution 1: 4) and incubated for 30 min at 37°. The cells were washed and resuspended in 1 ml of agaroseabsorbed guinea-pig serum (final dilution 1: 6). After incubation for an additional 30 min at 370, the cells were washed, and assayed for cell-mediated
cytotoxicity. Preparation ofstimulator cells 2 x 107 spleen cells depleted of erythrocytes as described elsewhere (Doherty & Zinkernagel, 1976) were incubated with a suspension of inactivated sendai virus containing 102_103 HAU in a total volume of 2 ml for 1 h at 37°. Thereafter the cells were X-irradiated with 2000 rad (Philips machine RT 200) and washed twice.
Measurement of delayed type hypersensitivity (DTH) to sendai virus Using the method of Tosolini & Mims (1971) mice were selected from the groups used experimentally and tested for DTH reactivity towards sendai viruses. In none of the CBA mice tested was a positive reactivity observed.
Virus specific CTL induced in vitro
765
Table 1. In vitro induction of sendai virus immune cytotoxic effector cells % Specific lysis of CBA macrophagest
Untreated Responder cells CBA CBA CBA
Stimulator* cells None CBA infected CBA treated with non-infectious virus§
Infected
Externally coated with inactivated virus§
t50:1
5:1
50: 1
5:1
50: 1
2 0
1 2
4 19
-1 11
6
6
2
61
47
50
5: 1 2 N.D. 24
* Ratio responder cells and stimulator cells was 6: 1. t Ratio effector cells to target cells. $ Background lysis of the target cells during the 3 h cytotoxicity assay was less than 34%4 § I-propionlactone-inactivated sendai virus.
RESULTS
Membrane-associated infectious sendai virus antigens induce virus specific CTL in vitro. There is evidence that sendai virus-specific CTL not only lyse sendai virus-infected syngeneic target cells but also syngeneic cells coated with non-infectious sendai virus particles (Schrader & Edelman, 1977; Koszinowski et al., 1977; Pfizenmaier et al., 1977a). Since these data suggested that inactivated virus material is sufficient to modify a cell and make it a specific 'target' for sendai virus immune CTL, we tested the possibility of using syngeneic cells coated with inactivated sendai virus in order to initiate in vitro primary anti-sendai virus CTL responses. For this purpose in a first step CBA mouse splenic lymphocytes were incubated in vitro with either infectious, or inactivated sendai virus for 60 min at 370 and washed (stimulator cells). In a second step CBA mouse splenic lymphocytes (4 x 106 responder cells) were co-cultured with 1 x 106 of stimulator cells. After 5 days the cultured cells were assayed for cytotoxicity towards sendai virus infected syngeneic macrophages. Representative results obtained are given in Table 1. Accordingly strong cytotoxicity could be generated using stimulator cells coated with inactivated sendai virus, whereas cultures containing sendai virus-infected stimulator cells generated only weak cytotoxicity. The data in Table 1 also indicate that sendai virus immune CTL generated in vitro lysed fl-propion lactone sendai virus treated target cells to a similar extent as target cells incubated with live virus. Thus both the afferent
limb and the efferent limb of a primary cytotoxic anti-sendai CTL response could be basically induced in vitro using inactivated sendai virus material.
Specificity of the cytotoxic activity generated Next, various parameters of the primary in vitro anti-sendai virus CTL response were defined. From the dose-response curve (Fig. 1) of stimulator cell formation using dilutions of 8-propionlactoneinactivated sendai virus, it can be deduced that about 100-1000 HAU equivalents of inactivated sendai virus material per 107 lymphocytes appeared to be optimal. Using stimulator cells obtained by treating CBA spleen cells (107) with 1000 HAU equivalents of inactivated sendai virus and titrating the optimal ratio of responder to stimulator cells for generating in vitro CTL responses, results as given in Fig. 2 were obtained. Accordingly, a ratio responder to stimulator cells of 4-8 to 1 yielded the highest CTL responses. Moreover, X-irradiating the stimulator cells made no obvious difference (Fig. 2). In Fig. 3 representative results of the kinetics of the induction of anti-sendai immune CTL are given. Similar to primary cytotoxic allograft reactions (Cerottini & Brunner, 1974; Wagner, Rollinghoff & Nossal, 1973), peak cytotoxic activity generated was observed at day 5 of culture. That the cytotoxic activity generated in vitro was due to T lymphocytes was strongly suggested by the observation that treatment of the effector cells with anti-Thy 1.2 serum plus complement abrogated their cytotoxic activity
(Table 2).
H. Jung et a!.
766 90
70(
70~~~ 70
aJ 4)
-30 3
*
i/
3 ___,_A_
10
.^A
AJI!
0
A
100
10
HA- titre
104
I0
ner 107 stimulotor
cells
Figure 1. Immunogenicity of syngeneic lymphocytes treated with a graded concentration of fl-propionlactone-inactivated sendai virus material. Splenic lymphocytes of CBA mice were divided into two parts. Aliquots (10' cells) of one part was incubated (30 min at 370) with a graded concentration of inactivated sendai virus (stimulator cells). The untreated cells (responder cells) were cultured together with the washed stimulator cells for 5 days. The cultured cells of the various groups were harvested and assayed for cytotoxicity in a 3 h 51Cr assay, towards CBA macrophages (A); sendai virus-infected CBA macrophages (0); and CBA macrophages treated with inactivated sendai virus (U) at an effector to target cell ratio of 50: 1. Background lysis of the targets was less than 31%.
80 r- (a) I
--
60 ~n -'
4
4 5 6 7 Days of culture
8
10
9
Figure 3. Kinetics of the generation of cytotoxicity. CBA splenic responder cells were cultured with A-propionlactoneinactivated sendai virus treated (103 HAU per 107 cells) CBA spleen cells (stimulator cells). At daily intervals groups of the cultures were harvested and assayed for cytotoxicity towards sendai virus-infected (0) or normal (A) CBA macrophages at an attacker to target cell ratio of 50: 1. Background lysis of the target cells during the 3 h assay was less than 31 %.
Table 2. Effect of anti-Thy 1.2 serum plus complement upon in vitro immunized cytotoxic lymphocytes
% Specific lysis of sendai virus-infected CBA-macrophages* Treatment of effector cells
50: 1
5: 1
None Anti-Thy 1.2 G.P. complement Anti-Thy 1.2 plus complement
78 77 70 9
49 42 25 7
* Ratio effector to target cells; assay time 3 h; background lysis 13 %.
40
20
1:1
4:1 8:1 40:1 1:1 4:1 8:1 Ratio resoonder to stimulator cells
40:1
Figure 2. Dose-response relation of cytotoxic activity generated in vitro: CBA splenic responder cells (4 x 106) were cultured with various numbers of stimulator cells (CBA spleen cells treated with 103 HAU inactivated sendai virus per 107 cells). At day 5 of culture the stimulated CBA cells were assayed in a 5'Cr-release test (3 h) towards sendai virus infected CBA macrophages. Background lysis of the target cells was less than 22%. (0) Ratio effector cells to target cells of 50: 1. (0) Ratio of 5: 1. (a) Non-irradiated stimulator cells; (b) X-irradiated (2500 rad) stimulator cells.
Parameters of primary in vitro anti-sendai virus CTL responses To test for the virus specificity of the CTL generated in vitro sensitized sendai immune CTL were tested for lytic activity towards syngeneic targets infected with either sendai virus, LCM virus or Herpes Type 1 virus (Pfizenmaier, Starzinski-Powitz, Rollinghoff, Falke & Wagner, 1977b). Similar to in vivo generated sendai immune CTL, in vitro induced sendai immune CTL exhibited a strict specificity for syngeneic targets infected with the virus used for the induction of the immune response (Table 3). In addition, in vitro generated sendai virus immune CTL did not
Virus specific CTL induced in vitro Table 3. Specificity of in vitro induced antisendai virus immune CTL
did not show any detectable DTH reactions to inactivated sendai virus (data not shown). To bypass virus-induced cytopathic effects within the cultures, we took advantage of the observation that non-infectious inactivated sendai virus particles are able to modify syngeneic cells and make them specific targets for sendai immune CTL (Pfizenmaier et al., 1977a; Schrader & Edelman, 1977; Koszinowski et al., 1977). Consequently we argued that syngeneic lymphocytes, the cell membrane of which had been fused with inactivated sendai virus particles may trigger in vitro primary sendai virus specific CTL responses. Indeed using both u.v. light irradiated or f,-propionlactone-inactivated sendai virus material at a concentration of about 100-1000 HAU per ml to treat 107 CBA splenic lymphocytes (stimulator cells), strong cytolytic activity towards both sendai virus infected and p8-propionlactoneinactivated sendai virus treated target cells was generated in vitro. Moreover, there was a clear doseresponse relationship between the concentration of inactivated sendai virus used for the treatment of stimulator cells and their immunogenicity (Fig. 1). In addition a critical ratio of responder to stimulator cells was required in order to induce efficient sendai virus-specific CTL (Fig. 2). Finally the CTL generated in vitro exhibited the same features as in vivo sensitized CTL, that is they were virus specific and H-2 restricted. It is concluded therefore that the in vitro approach described results in the induction of virus specific CTL functionally similar to those activated under in vivo conditions. Thus the system may be of advantage in dissecting and analysing the sensitization phase of CTL precursor cells reactive with specific viral antigens on H-2 restricted target
% Specific lysis of CBA macrophages Virus strain used for infection of targets None LCM Herpes Typ 1 Sendai
50: 1*
5: 1
4 3 -2 70
2 0 1 29
767
* Ratio of in vitro sensitized CBA anti-sendai virus immune CTL (attacker cells) to target cells. Background lysis of the target cells was less than 32 %.
lyse allogeneic target cells infected with sendai virus (Table 4) thus demonstrating the same H-2 restriction as in vivo induced virus immune CTL (Doherty & Zinkernagel, 1976; Pfizenmaier et al., 1977a; Ertl & Koszinowski, 1976). DISCUSSION The aim of the experiments reported was to induce in vitro a primary T-cell-mediated cytotoxic immune response to sendai virus infected cells. To exclude the possibility that the mice used in the experiments had been already in contact with sendai virus and that therefore we would be dealing with an in vitro sensitization of in vivo primed T lymphocytes, the CBA mice used experimentally were killed within 10 days of removing them from SPF conditions. Such mice
Table 4. H-2 restriction of in vitro sensitized sendai virus specific CTL
% Specific lysis of sendai virus-infected macrophages CBA
Sendai virus specificCTL* CBA
B1O(4R) B1O.D2
BALB/c
BlO
BlO.A
B1O(4R)
t50: 1 5:1 50:1 5:1 50:1 5: 1 50:1 5:1 50:1 5:1 48 19 5
12 5 2
9 0 77
6 1 18
1 0 0 -2 N.D.
32 13 34
15 6 3
33 6 13 2 N.D.
* In vitro sensitization was performed as under conditions described in Table 1. t Ratio of attacker to target cells used in the 3 h cytotoxicity assay. Each effector cell population was tested in an independent assay against the target cells listed. Background lysis of the target cells was less than 26 %.
768
H. Jung et al.
cells. The data indicate that, for the induction of sendai virus-specific CTL, no translation of early viral proteins nor expression of viral proteins on the cell surface from 'within' is required. This is in contrast to results obtained in the vaccinia system (Ada, Jackson, Blanden, Tha Hla & Bowern, 1976). Most likely the capacity of inactivated sendai virus to bypass these requirements is due to its unique capacity to mediate cell-cell fusion, which in turn allows by mere incubation with inactivated sendai viruses the insertion of large amounts of virus material into the membrane of both stimulators and target cells. If so, one may deduce that virus-specific CTL can be generated in vitro against a variety of antigens, provided they can be inserted properly into the cell membrane of the stimulator cells and target cells. Various laboratories have succeeded inducing in vitro secondary sendai virus-specific CTL responses (Schrader & Edelman, 1977; Koszinowski et al., 1977; Zweerink, Courtneidge, Skehel, Crumpton & Askonas, 1977). In one report (Schrader & Edelman, 1977) small but significant primary sendai virus specific CTL responses have been described. By analysing the various parameters required for the in vitro induction of virus-specific CTL, we now report cytolytic activity comparable to those obtained by an in vivo immunization regime (Doherty & Zinkernagel, 1976; Ertl & Koszinowski, 1976). Of special interest is the simplicity by which virus-specific CTL can be induced and assayed in a short termed 3 h 51Cr-test. We therefore hope that the in vitro approach described may be useful in analysing the cellular events resulting in vitro in H-2 restricted primary induced virus-specific CTL.
ACKNOWLEDGMENTS The skilled technical assistance of Mrs A. Neumann is gratefully acknowledged. This work was supported by the SFB 107.
REFERENCES ADA G.L., JACKSON D.C., BLANDEN R.V., THA HLA R. & BOWEN N.A. (1976) Changes in the surface of virusinfected cells recognized by cytotoxic T cells. I. Minimal
requirements for lysis of ectromelia-infected P-815 cells. Scand. J. Immunol. 5, 23. BLANDEN R.V., PANG T.E. & DUNLOP M.B.C. (1977) T cell recognition of virus-infected cells. In: Cell surface reviews,
vol. 2 (Ed. by G. Poste and G. L. Nicolson), p. 249. North Holland, Amsterdam. CERorrINI J.C. & BRUNNER K.T. (1974) Cell-mediated cytotoxicity, allograft rejection, and tumor immunity. Adv. Immunol. 18, 67. DOHERTY P.C., ZINKERNAGEL R.M. & RAMSHAW I.A. (1974) Specificity and development of cytotoxic thymus-derived lymphocytes in lymphocytic choriomeningitis. J. Immunol. 112, 1548. DOHERTY P.C., GOTZE D., TRINCHIERI G. & ZINKERNAGEL R.M. (1976) Models for recognition of virally modified cells by immune thymus-derived lymphocytes. Immunogenetics, 3, 517. DOHERTY P.C. & ZINKERNAGEL R.M. (1976) Specific immune lysis of paramyxovirus-infected cells by H-2 compatible thymus-derived lymphocytes. Immunology, 31, 27. ERTL H. & KosZINOWSKI U. (1976) Cell-mediated cytotoxicity against sendai-virus-infected cells. Z. ImmunForsch. 152, 128. JANEWAY C.A. Jr, WIGZELL H. & BINz H. (1976) Two different VH gene products make up the T-cell receptors. Scand. J. Immunol. 5, 993. KosZINOWSKI U. & ERTL H. (1975) Lysis mediated by T cells and restricted by H-2 antigen of target cells infected with vaccinia virus. Nature (Lond.), 255, 552. KOsZINOWSKI U., GETHING M.J. & WATERFIELD M. (1977) T-cell cytotoxicity in the absence of viral protein synthesis in target cells. Nature (Lond.), 267, 160. PFIZENMAIER K., TROSTMANN H., ROLLINGHOFF M. & WAGNER H. (1976a) Cell-mediated immunity in lymphocytic choriomeningitis. I. The specificity of the cytotoxic T lymphocytes. Z. ImmunForsch. 151, 224. PFIZENMAIER K., STARZINSKI-POWITz A., RODT H., ROLLINGHOFF M. & WAGNER H. (1976b) Virus and trinitrophenyl hapten-specific T-cell-mediated cytotoxicity against H-2 incompatible target cells. J. exp. Med. 143, 999. PFIZENMAIER K., STARZINSKI-POWITz A., WAGNER H. & ROLLINGHOFF M. (1977a) T-cell-mediated antiviral cytotoxicity: Cell membrane associated virion antigen is sufficient for lysis of H-2 matched target cells. Z. ImmunForsch. 153, 268. PFIZEMAIER K., STARZINSKI-POWITZ A., ROLLINGHOFF M., FALKE D. & WAGNER H. (1977b) T-cell-mediated cytotoxicity against herpes simplex virus-infected target cells. Nature (Lond.), 265, 630. SCHRADER J.W. & EDELMAN G.M. (1977) Joint recognition by cytotoxic T cells of inactivated sendai virus and products of the major histocompatibility complex. J. exp. Med. 145, 523. TOSOLINI F.A. & MiMs C.A. (1971) Effect of murine strain and viral strain on the pathogenesis of lymphocytic choriomeningitis infection and a study of footpad responses. J. Infect. Dis. 123, 134. WAGNER H., GOTZE D., PTSCHELINZEW L. & ROLLINGHOFF M. (1975) Induction of cytotoxic T lymphocytes against I-region-coded determinants: in vitro evidence for a third histocompatibility locus in the mouse. J. exp. Med. 142, 1477. WAGNER H., ROLLINGHOFF M. & NOSSAL G.J.V. (1973) T-cell-mediated immune responses induced in vitro: A probe for allograft and tumor immunity. Transplant. Rev. 17, 3.
Virus specific CTL induced in vitro WAGNER H. & ROLLINGHOFF M. (1974) T-cell-mediated cytotoxicity: discrimination between antigen recognition, lethal hit and cytolysis phase. Eur. J. Immunol. 4, 475. ZINKERNAGEL R.M. (1976) H-2 restriction of virus-specific cytotoxicity across the H-2 barrier. Separate effector T cell specificities are associated with self-H-2 and with the
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tolerated allogeneic H-2 in chimeras. J. exp. Med. 144, 933. ZWEERINK H.J., COURTNEIDGE S.A., SKEHEL J.J., CRUMPTON M.J. & ASKONAS B.A. (1977) Cytotoxic T cells kill influenza virus infected cells but do not distinguish between serologically distinct type A viruses. Nature (Lond.), 267, 354.
