Clin. exp. Immunol. (1978) 31, 209-217.
Cytostasis against self-antigens LUISA DE GIORGI, ARPI MATOSSIAN-ROGERS & HILLIARD FESTENSTEIN Department of Immunology, The London Hospital Medical College, Turner Street, London
(Received 28 July 1977) SUMMARY
Effector cells from mice of the C57B11/10 congenic series, alloimmunized against K end- or D end-identical I region incompatible lymphocytes, were cytostatic for tumour and macrophage targets bearing syngeneic K-end and D-end antigens. If incompatibility during immunization was restricted to the I-B subregion only, cytostasis was directed against K end syngeneic antigens; further incompatibility in the I region was required to obtain cytostasis against D-end self-
antigens. Cytostasis against self-antigens was also obtained after immunization with syngeneic tumour cells; in one instance this was directed against syngeneic K-end antigens only. The implications of these findings in relation to neoplastic change and autoimmune damage are discussed. INTRODUCTION The immune response against syngeneic tumours has been assayed in vitro by a variety of techniques involving cytolysis and/or growth inhibition of tumour target cells (Hellstr6m et al., 1971; Finkelstein et al., 1972; Evans et al., 1972; Brunner & Cerottini, 1971). The existence of at least two types of effector systems in anti-tumour immunity was recognized by Lamon et al. (1973) and Plata et al. (1974) in a cell-mediated microcytotoxicity assay (MA), using monolayers of MSV tumour cells. The growth inhibitory component could be abrogated by serum from tumour-bearing mice and soluble tumour antigens (Hellstrom & Hellstrom, 1969; Plata & Levy, 1974), or antigen-antibody complexes (Sjdgren et al., 1971), without affecting the cytolytic potential of the effector cells against target cells in suspension (Leclerc et al., 1973; Plata et al., 1973). The in vitro conditions to assay growth inhibition of tumour cells termed 'cytostasis' were developed by Chia & Festenstein (1973) to study the anti-tumour immune reaction in mice bearing autochthonous chemically induced tumours, and were also used by Senik, De Giorgi & Levy (1974a) for virally induced tumours. The type of effector cell in cytostasis is still unknown, but does not appear to be a T cell. Senik et al. (1974b) attributed the effector function to macrophages and B cells and showed that specificity was only achieved at low effector/target cell ratios. Large numbers of effector cells per target do not affect the specificity of cell-mediated cytolysis, which is a pure T-cell function. The involvement of antigen-antibody complexes in the mechanism of MA was shown by Sjogren et al. (1971) and Baldwin et al. (1973), and recent findings by Farram & Festenstein (1977), using an immunofluorescence technique, support the idea that cytostasis is due to a specific antibody reaction against tumour cells. The role of the H-2 complex in the sensitization phase and in encoding for targets in cytostasis has already been demonstrated using H-2 congenic and recombinant mice for alloimmunization and tumour cells of different haplotypes as targets (De Giorgi, Biasi & Festenstein, 1976). To generate cytostatic effector cells an I-B region incompatibility was essential and this presented with K-end or D-end differences directed specificity against these alloantigens. For example, B1O.A(5R) anti-B10.A effector Correspondence: Dr Luisa De Giorgi, Department of Immunology, The London Hospital Medical College, Turner Street, London El 2AD. 0099-9104/78/0200-0209$02.00 ©1978 Blackwell Scientific Publications 209
210
Luisa De Giorgi, Arpi Matossian-Rogers C Hilliard Festenstein
cells (bbbddd anti-kkkddd) were strongly cytostatic for Gardner tumour targets (H-2k) but not for MBL2 (H-2b) or SL2 (H-2d). Similarly, B10.A anti-B1O.A(4R) effectors (kkkddd anti-kkbbbb) were cytostatic for MBL2 (H-2b) but not for the H-2k and the H-2d tumours. A combination, however, which did not give rise to an I-B region incompatibility B1O.A(5R) anti-B1O.A(4R) (bbbddd anti-kkbbbb) was incapable of producing cytostasis against any of the tumour targets (De Giorgi, Biasi & Festenstein, 1977). In this report we show that I-B region incompatibility alone is able to activate an immune response to self-antigens which is normally repressed and that the targets in syngeneic cytostasis are encoded by K-end and D-end regions. Using syngeneic tumour cells for immunization, cytostatic effector cells were also produced with specificity for K- or D-end antigens on syngeneic and other tumours.
MATERIALS AND METHODS Mice and tumours. For the production of effector cells in a cytostasis assay the following inbred strains of mice were used: BALB/c (H-2d), A/Jax (H-2a), congenic mice of the C57BI/10 series and certain F1 hybrids between these congenic strains. Tumours used as targets in the cytostasis assay and for in vivo immunizations were maintained in ascites in their strains of origin by weekly passage of 2x 106 tumour cells injected intraperitoneally (i.p.). These are listed in Table 1. Immunizations. Mice were injected i.p. with 10' allogenic spleen cells from the appropriate strains. Tumour-bearing mice were produced by intramuscular (i.m.) injection of 106 tumour cells into one hind leg. Cytostasis assay. Lymph nodes were removed from immunized mice between 10 to 15 days after immunization and single cell suspensions were prepared in a mixture of RPMI 1640 and MEM (Wellcome) supplemented with 5% foetal calf serum (Gibco). Tumour targets were obtained from the ascites of the appropriate mouse strain, washed twice and suspended in the above medium. Allogeneically or syngeneically immunized effector cells were mixed with tumour targets at ratios of 5:1 and 20:1, respectively, in a total volume of 1-0 ml in Greyward 1158 culture tubes. Lymphocytes from normal mice were mixed at the same ratios with tumour targets for control purposes. The tubes were incubated at 37°C for 48 hr in a 5% CO2 atmosphere. During the last 16 hr they were pulsed with [14C]thymidine. The cells were harvested on fibre filter strips using a semi-automatic harvesting machine and the isotope uptake was determined using a liquid scintillation spectrometer. In preliminary experiments it was shown that the uptake of isotope by the immune (IL) or normal (NL) lymphocytes was negligible during this period of incubation (Chia & Festenstein, 1973), thus the counts were taken to represent the proliferation of tumour cells. Cytostasis of tumour cells was expressed as a percentage inhibition of growth calculated according to the following formula (Senik et al., 1974a): percentage inhibition = 1000
tumour cells+IL -t/min ofof normal cells+NL
ct/min
Metabolic inhibition (cytostasis) assay using macrophage targets. This was carried out as previously described (MatossianRogers & Festenstein, 1977). Briefly, 5 x 104 peritoneal macrophages in culture medium were dispensed in each well of flat-bottom Microtest II plates (Cooke N/220-29AR, Cooke Laboratory Products, Alexandria, Virginia), incubated for a minimum of 2 hr at 37°C in a 5% CO2 atmosphere and washed to remove non-adherent cells just before use. TABLE 1. Tumour target cells: induction and strains of origin
Tumour cells
MBL2 (leukaemia) Gil4 (lymphoma) SL2 (lymphoma) LSTRA (sarcoma) P815-y (mastocytoma) Meth.A (lymphosarcoma) Gardner (lymphosarcoma) YAC (lymphoma) 4RLM (leukaemia)* *
Induction
Moloney virus Graffi virus Spontaneous Moloney virus
Methylcholanthrene Methylcholanthrene Oestrogen Moloney virus Moloney virus
Strains of
H-2
origin
haplotype
C57BI/6 C57BI/6 DBA/2 BALB/c DBA/2 BALB/c CBA/H A/Jax B1OA. (4R)
The 4RLM tumour was induced in our laboratory by Dr Giovanni Biasi.
H-2b H-2b
H-2d H-2d H-2d H-2d H-2k H_2a H-2h4
Cytostasis against self-antigens
211
Equal volumes of responder and irradiated stimulator lymphocytes at 2-5 x 106 per ml were cultured for 4 days; effector cells were removed and overlayed on macrophage targets at a ratio of 5 1. Normal cells were used at the same ratio for control purposes. The plates were incubated for 48 hr at 37'C in a 5%Y CO2 atmosphere. The effector cells were then washed off and 200 pl of culture medium together with 10pl of 14Clabelled uridine (0-33 pCi/mIl) were added to each well. The uptake of isotope during a further 18 hr incubation period was measured. The inhibition of isotope uptake was calculated using the formula: ct/min of macrophages + IL 1 100. percentage inhibition = 100- crp ct/min of macrophages+NLj Experimental design. Our previous experiments, using alloimmune effector cells across H-2 region incompatibilities demonstrated that cytostasis against H-2 coded alloantigens on tumour cells could be achieved if H-2K or H-2D region differences were presented together with an I-B region incompatibility during the in vivo sensitization. In the present experiments, we first showed that cytostasis occurred against syngeneic targets using effector cells from mice immunized with I-B region incompatible cells. To examine the targets of this cytostasis, combinations of B10 congenic mice and selected F1 hybrids were used to obtain incompatibilities in the I region alone. These combinations with I region differences only are henceforth termed 'syngeneic'. Cytostasis was achieved against tumour targets sharing the K- or D-end antigens of the responder cells. To confirm that cytostasis was directed against self-antigens, macrophages syngeneic to the responder lymphocytes were also used as targets in a post-labelling assay measuring 14C-labelled uridine uptake. This assay detected diminished metabolic activity of the target cells after treatment with immune cells as compared to normal cells, and is probably mediated by mechanisms similar to those that effect the stasis of tumour cell proliferation. Further experiments examined the specificity of cytostasis in immunizations with syngeneic tumour cells.
RESULTS A11oimmunization can induce 'syngeneic' cytostasis Specific cytostasis against self-antigens expressed by K-end genes of the H-2 complex is obtained by immunizing mice with an effective I-B region incompatibility only. Table 2 shows several combinations where immunization across this restricted incompatibility is achieved by the use of F1 hybrids as responders. Effector cells obtained from [B1O.A(4R) x BIO.A(5R)]F1 mice immunized against B1O.A (I-B region incompatibility) were cytostatic for tumour cells bearing syngeneic K-end specificities (Gardner) but not syngeneic D-end specificities (MBL2). This represents 'syngeneic' cytostasis, since the responder strain also expressed H-2k antigens at the K end. Two further combinations [B1O.A(4R) x B1O.D2]F1 immunized against B1O.A(2R) and [B1O.A(4R) x B1O.D2]F1 immunized against B1O.A yielded cytostatic effector cells for tumours expressing syngeneic K-end specificities. It is unlikely that the cytostasis in these combinations is directed against the I-B region specificities. B1O.A(4R) anti-B1O.A(2R) effector cells, which represent b anti-k at the I-B region, were cytostatic for tumour cells carrying both K-end (H-2k, Gardner) and D-end (H-2b, Gil 4) specificities of the effector cells. The D end in this combination bears no apparent relationship to the specificity of the I-B region immunization. An analogous situation was noted with B1O.A(2R) anti-B1O.A(4R) effector cells, where cytostasis of the H-2k tumour (Gardner) did not follow the specificity of the I-B region incompatibility, which was k anti-b (Table 2). If the incompatibility between BlO.A(4R) and B1O.A(2R) was narrowed down to the I-B region only by immunizing a [B1O.A(4R) x B1O.D2]F1 hybrid against B1O.A(2R), then cytostasis was directed only against tumours sharing K-end antigens of the effector cells. It can be concluded from these experiments that an I-B region incompatibility generates cytostasis against K-end syngeneic targets, irrelevant of the specificity of the I-B region immunization, and additional I region incompatibility is necessary to obtain cytostasis to D-end targets. Cytostasis using macrophage targets The cytostatic potential of effector cells immunized against I region incompatibilities alone was tested against macrophage targets in a metabolic inhibition assay. B1O.A(2R) anti-B1O.A(4R) effector cells were strongly cytostatic for B1O.A and B10 macrophages
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which share K-end and D-end specificities, respectively, of the responder strain, and also for syngeneic macrophages and B10.A(4R) macrophages, which are identical at the K end and D end with the responder lymphocytes (Table 3). When B1R.A was immunized against B1O.A(4R), providing I region and D-end incompatibilities, cytostasis was directed against the H-2b alloantigen (B10 macrophages) and also against self-antigens expressed on syngeneic (B1O.A) and allogeneic macrophages [B1O.A(4R) and B1O.A(2R)]. These results demonstrate that the targets for cytostasis in combinations incompatible for I region alone are selfantigens.
Cytostasis against syngeneic tumours Immunization of pure inbred or recombinant mice against syngeneic tumours resulted in the generation of cytostatic effector cells against syngeneic tumour targets of the pure haplotype or K-end and D-end specificities of the recombinant haplotypes. Effector cells from B1O.A(4R) mice immunized with syngeneic tumour cells (4RLM) (Table 4) were cytostatic for YAC and Gardner tumours syngeneic with the effector cells at the K end and also for MBL2 syngeneic at the D end. BALB/c mice immunized against the syngeneic Meth.A tumour were cytostatic only for H-2d-bearing targets, such as YAC and Meth.A, and not for H-2b (MBL2) or H-2k (Gardner). Immunization of A/Jax (H-2a) and B10.A (H-2a) mice with YAC tumour syngeneic to A/Jax resulted in an interesting difference. A/Jax effector cells were cytostatic to both K-end and D-end targets, while B10.A effector cells were cytostatic to K-end targets only. The restricted effector capacity in the latter case may be explained in the light of the previous data in that YAC tumour cells may have presented a narrower range of incompatibility to B10.A responder lymphocytes than to A/Jax lymphocytes. This will be further discussed. DISCUSSION Virally or chemically induced tumours carry transplantation antigens which stimulate the development of effector cells in syngeneic hosts. The nature of these transplantation antigens is still unclear; however, they induce or become targets for cytotoxic cells (Brunner & Cerottini, 1971) or, as we have demonstrated in this report (Table 4), for cytostatic effector cells. These two immune phenomena are distinct in their mechanism and even though they may coexist in vivo, they are separable in vitro (Plata et al., 1974; Seeger & Owen, 1973). We have previously shown that to generate cytostatic effector cells directed against MHC gene products, an I-B region incompatibility between responder and stimulator lymphocytes is essential (De Giorgi et at., 1977). Cytotoxic cells, however, can be produced in I-B region compatible combinations of recombinant C57B1/10 congenic mice (Matossian-Rogers, Demant & Festenstein, 1975). This is an important difference in the sensitization phase of the two effector systems. If I-B region incompatibility is presented together withH-2K or H-2D region differences, thencytostasis against these alloantigens on tumour targets is achieved. If I-B region incompatibility is presented alone, cytostasis still occurs and is directed against syngeneic K-end antigens only, while a broadening of the I region incompatibility extends the specificity of syngeneic cytostasis to D-end targets as well. Brondz, Egorov & Drizlikh (1975) showed that different clones of T cells are specific for H-2K or H-2D antigens. It is possible that clones of lymphocytes with helper or effector function with restricted specificity may be differentially activated by various regions of the H-2 complex. To explain the generation of cytostasis against syngeneic tumour targets on the basis of the incompatibility presented by TSTA, we postulate that the viral or chemical induction of tumours may be associated with an I region modification of the neoplastic tissue. The extent of this modification then determines whether cytostasis will be directed to K-region or D-region antigens. The differential response of A/Jax and B10.A may be explained by this hypothesis, if we assume that they have minor I region differences (hitherto undetected) and that the modified I region of the YAC
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tumour is more similar to the I region of the B1O.A mouse. As a corollary, these results would then be the first indication of I region differences in A/Jax and B1O.A mice! The nature of the I region modification which initiates an immune response to self-antigens is not clear. Though it may be a serologically detectable new Ia specificity, and Ia specificities of foreign haplotypes have been found on SL2 tumour cells (Garrido, Festenstein & Schirrmacher, 1976; Hammerling et al., 1974), no known Ia specificity has yet been definitively mapped in the I-B region (Shreffler, Meo & David, 1977), suggesting that it is more likely to give rise to a cell-mediated response. Evidence that tumour-specific transplantation antigens are modified histocompatibility antigens has been previously demonstrated by Bowen & Baldwin (1975). The question still remains how a modification in the I region breaks the tolerance to self-antigens and makes them targets for immune effector cells. Since cytostasis is directed against K- or D-region antigens on autochthonous and other syngeneic tumours (Table 4), the specificity of these gene products does not appear to be altered by the oncogenic agent. Nevertheless, allosteric alteration, possibly by mechanical distortion, may take place without affecting specificity, thus providing a new acceptor site for recognition structures on immunocompetent cells. This may be analogous to some interactions of virus-immune lymphocytes against syngeneic virustransformed target cells for viruses that may not cause any serologically detectable modification of the cell membrane (Trinchieri, Aden & Knowles, 1976). On the other hand, no alteration in structure may be necessary if a two-receptor hypothesis is invoked for self-recognition (Doherty et al., 1976). The V-region genes may be phenotypically expressed on the lymphocyte membrane, one recognizing H-2K or H-2D and the other possibly an I region modification. Recognition by one signal alone, for example the idiotype for H-2K, may not provide sufficient avidity for the effector phase, while multiple recognition by two or more idiotypic receptors would achieve the threshold for immunological function. Such a system would be compatible with any mechanism that may effect cytostasis, since both T and B lymphocytes directed against the same alloantigens have been shown to use receptors with shared idiotypic determinants (Binz & Wigzell, 1975a,b). Finally, our results may shed some light upon self-recognition mechanisms in autoimmune phenoma. Genes mapping at H-2K or H-2D function as self-markers in cell-mediated immunity, as shown in systems using virus-infected syngeneic cells. The classical HLA antigens in man may have similar functions, while modification of antigens analogous to those encoded by the I region, possibly the B lymphocyte antigens, may be the triggering mechanism for autoimmune damage. We thank Dr Giovanni Biasi for inducing the 4RLM tumour and the Cancer Research Campaign for financial support.
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BINz, H. & WIGZELL., H. (1975a) Shared idiotypic determinants on B and T lymphocytes reactive against the same antigenic determinants. I. Demonstration of similar or identical idiotypes on IgG molecules and T-cellreceptors with specificity for the same alloantigens. 3. exp. Med. 142, 197. BINZ, H. & WIGZELL, H. (1975b) Shared idiotypic determinants on B and T lymphocytes reactive against the same antigenic determinants. II. Determination of frequency and characteristics of idiotypic T and B lymphocytes in normal rats using direct visualization 3. exp. Med. 142,1218. BowEN, J.G. & BALDWIN, R.W. (1975) Tumour-specific antigen related to rat histocompatibility antigens. Nature (Lond.), 258, 75. BRONDZ, B.D., EGOROV, I.K. & DRIZLIKH, G.I. (1975) Private specificities of H-2K and H-2D loci as possible selective targets for effector lymphocytes in cell-mediated immunity. J. exp. Med. 141, 11.
BRUNNER, K.T. & CEROTTINI, J.C. (1971) Cytotoxic lymphocytes as effector cells of cell-mediated immunity. Progress in Immunology, p. 385. Academic Press, London and New York. CHIA, E. & FESTENSTEIN, H. (1973) Specific cytostatic effect of lymph node cells from normal and T-cell deficient mice on syngeneic tumour target cells in vitro, and its specific abrogation by syngeneic fluid from tumourbearing mice. Europ. 1. Immunol. 3, 483. DE GIORGI, L., BIASI, G. & FESTENSTEIN, H. (1976) The genetics of cytostasis. Folia biol. (Praha), 22, 437. DE GIORGI, L., BIAsI, G. & FESTENSTEIN, H. (1978) The role of H-2 and H-2-like determinants in syngeneic and allogeneic cytostasis. J. Immunogenet. 5, 49. DOHERTY, P.C., GOTZE, D., TRINCHIERI, G. & ZINKERNAGEL, R.M. (1976) Hypothesis. Models for recognition of virally modified cells by immune thymus-derived lymphocytes. 5. Immunogenet. 3, 517. EvANs, R. & ALEXANDER, P. (1972) Mechanism of
Cytostasis against self-antigens immunologically specific killing of tumour cells by macrophages. Nature (Lond.), 236, 168. FARRAM, E. & FESTENSTEIN, H. (1977) The role of antibody against tumour antigens in the inhibition of tumour growth in vivo and in vitro. (In press.) FINKELSTEIN, J.Z., BrYFIW, J., TITmE, K. & IMAGAWA, D.T. (1972) Studies of inhibition of tumour-cell DNA synthesis by immune cells in Gross virus induced leukaemia. Rev. Europ. Etud. Clin. Biol. 17, 287. GARRiDo, F., FEsTENsTEIN, H. & SCIRRMACHER, V. (1976) Further evidence for derepression of H-2 and Ia-like specificities of foreign haplotypes in mouse tumour cell lines. Nature (Lond.), 261, 705. HAMMERLING, G., DEAK, B., MAUVE, G., HAMMRLING, V. & McDEVITT, H.O. (1974) B lymphocyte alloantigens controlled by the I region of the major histocompatibility complex in mice. 3. Immunogenet. 1, 68. HELLSTROM, I. & HELLSTROM, K.E. (1969) Studies on cellular immunity and its serum-mediated inhibition in Moloney virus induced mouse sarcoma. Int. 3. Cancer, 4, 587. HELLSTROM, K.E., HELLSTROM, I., SjOGREN, H.O. & WARNER, G.A. (1971) Progress in Immunology (ed B. Amos), Vol. 1, 939. LAMON, E.W., WIGZELL, H., KLEIN, E., ANDERSON, B. & SKURZAK, H.M. (1973) The lymphocyte response to a primary Moloney sarcoma virus induced tumour in BALB/c mice: definition of the active subpopulations at different times after infection. 3. exp. Med. 137, 1472. LECLERC, J.C., GomARD, E., PLATA, F. & LEVY, J.P. (1973) Cell-mediated immune reaction against tumours induced by oncornaviruses. II: Nature of the effector cells in tumour cell cytolysis. Int. J. Cancer, 11, 426. MATOSSIAN-ROGERS, A., DEmANT, P. & FEsuENsTEIN, H. (1975) Further studies on the genetic basis of the generation of CML in mice. 3. Immunogenet. 2, 291. MATossIAN-RoGERS, A. & FEsTENsTEIN, H. (1977) Cytostatic effector cells generated in vivo against M locus determinants. Clin. exp. Immunol. 27, 335.
217
PLATA, F., GOMARD, E., LECLERC, J.C. & LEVY, J.P. (1973) Further evidence of the involvement of thymus-processed lymphocytes in syngeneic tumour cell cytolysis. 1. Immunol. 3, 667. PLATA, F. & LEVY, J.P. (1974) Comparative studies on the blocking of syngeneic effector T cells by soluble tumour antigens. Nature (Lond.), 249, 271. PLATA, F., GoMARD, E., LECLERC, J.C. & LEVY, J.P. (1974) Comparative in vitro studies on effector cell diversity in the cellular immune response to murine sarcoma virus (MSV) induced tumours in mice. .. Immunol. 112, 4. SENIK, A., DE GIORGI, L. & LEVY, J.P. (1974a) Cellmediated antitumour immunity in oncornavirus-induced tumours: specific cytostasis of tumour cells by spleen and lymph node cells. Int. J. Cancer, 14, 386. SENIK, A., DE GIORGI, L., GoMAiW, E. & LEVY, J.P. (1974b) Cytostasis of lymphoma cells in suspension: probable non-thymic origin of the cytostatic lymphoid cells in mice bearing MSV-induced tumours. Int. . Cancer, 14, 396. SEEGER, R.C. & OwEN, J.J.T. (1973) Measurement of tumour immunity in vitro with 125I-iododeoxyuridinelabelled target cells. Transplantation, 15, 404. SHREFFLER, D.C., MEo, T. & DAVID, C.S. (1977) Genetic resolution of the products and functions of I and S region genes of the mouse H-2 complex. The Role of Products of the Histocompatibility Gene Complex In Immune Responses (eds D. Katz and B. Benacerraf), p.3. Academic Press, New York. SJOGREN, H.O., HELLSTROM, I., BANSOL, S.C.,& HIELLSTROM, K.E. (1971) Suggestive evidence that the 'blocking antibodies' of tumour bearing individuals may be antigenantibody complexes. Proc. nat. Acad. Sci. (Wash.), 68, 1372. TRINCHIERI, G., ADEN, D. & KNowLES, B.B. (1976) Cellmediated cytotoxicity to SV40 specific tumour-associated antigens. Nature (Lond.), 261, 312.
Cytostasis against self-antigens LUISA DE GIORGI, ARPI MATOSSIAN-ROGERS & HILLIARD FESTENSTEIN Department of Immunology, The London Hospital Medical College, Turner Street, London
(Received 28 July 1977) SUMMARY
Effector cells from mice of the C57B11/10 congenic series, alloimmunized against K end- or D end-identical I region incompatible lymphocytes, were cytostatic for tumour and macrophage targets bearing syngeneic K-end and D-end antigens. If incompatibility during immunization was restricted to the I-B subregion only, cytostasis was directed against K end syngeneic antigens; further incompatibility in the I region was required to obtain cytostasis against D-end self-
antigens. Cytostasis against self-antigens was also obtained after immunization with syngeneic tumour cells; in one instance this was directed against syngeneic K-end antigens only. The implications of these findings in relation to neoplastic change and autoimmune damage are discussed. INTRODUCTION The immune response against syngeneic tumours has been assayed in vitro by a variety of techniques involving cytolysis and/or growth inhibition of tumour target cells (Hellstr6m et al., 1971; Finkelstein et al., 1972; Evans et al., 1972; Brunner & Cerottini, 1971). The existence of at least two types of effector systems in anti-tumour immunity was recognized by Lamon et al. (1973) and Plata et al. (1974) in a cell-mediated microcytotoxicity assay (MA), using monolayers of MSV tumour cells. The growth inhibitory component could be abrogated by serum from tumour-bearing mice and soluble tumour antigens (Hellstrom & Hellstrom, 1969; Plata & Levy, 1974), or antigen-antibody complexes (Sjdgren et al., 1971), without affecting the cytolytic potential of the effector cells against target cells in suspension (Leclerc et al., 1973; Plata et al., 1973). The in vitro conditions to assay growth inhibition of tumour cells termed 'cytostasis' were developed by Chia & Festenstein (1973) to study the anti-tumour immune reaction in mice bearing autochthonous chemically induced tumours, and were also used by Senik, De Giorgi & Levy (1974a) for virally induced tumours. The type of effector cell in cytostasis is still unknown, but does not appear to be a T cell. Senik et al. (1974b) attributed the effector function to macrophages and B cells and showed that specificity was only achieved at low effector/target cell ratios. Large numbers of effector cells per target do not affect the specificity of cell-mediated cytolysis, which is a pure T-cell function. The involvement of antigen-antibody complexes in the mechanism of MA was shown by Sjogren et al. (1971) and Baldwin et al. (1973), and recent findings by Farram & Festenstein (1977), using an immunofluorescence technique, support the idea that cytostasis is due to a specific antibody reaction against tumour cells. The role of the H-2 complex in the sensitization phase and in encoding for targets in cytostasis has already been demonstrated using H-2 congenic and recombinant mice for alloimmunization and tumour cells of different haplotypes as targets (De Giorgi, Biasi & Festenstein, 1976). To generate cytostatic effector cells an I-B region incompatibility was essential and this presented with K-end or D-end differences directed specificity against these alloantigens. For example, B1O.A(5R) anti-B10.A effector Correspondence: Dr Luisa De Giorgi, Department of Immunology, The London Hospital Medical College, Turner Street, London El 2AD. 0099-9104/78/0200-0209$02.00 ©1978 Blackwell Scientific Publications 209
210
Luisa De Giorgi, Arpi Matossian-Rogers C Hilliard Festenstein
cells (bbbddd anti-kkkddd) were strongly cytostatic for Gardner tumour targets (H-2k) but not for MBL2 (H-2b) or SL2 (H-2d). Similarly, B10.A anti-B1O.A(4R) effectors (kkkddd anti-kkbbbb) were cytostatic for MBL2 (H-2b) but not for the H-2k and the H-2d tumours. A combination, however, which did not give rise to an I-B region incompatibility B1O.A(5R) anti-B1O.A(4R) (bbbddd anti-kkbbbb) was incapable of producing cytostasis against any of the tumour targets (De Giorgi, Biasi & Festenstein, 1977). In this report we show that I-B region incompatibility alone is able to activate an immune response to self-antigens which is normally repressed and that the targets in syngeneic cytostasis are encoded by K-end and D-end regions. Using syngeneic tumour cells for immunization, cytostatic effector cells were also produced with specificity for K- or D-end antigens on syngeneic and other tumours.
MATERIALS AND METHODS Mice and tumours. For the production of effector cells in a cytostasis assay the following inbred strains of mice were used: BALB/c (H-2d), A/Jax (H-2a), congenic mice of the C57BI/10 series and certain F1 hybrids between these congenic strains. Tumours used as targets in the cytostasis assay and for in vivo immunizations were maintained in ascites in their strains of origin by weekly passage of 2x 106 tumour cells injected intraperitoneally (i.p.). These are listed in Table 1. Immunizations. Mice were injected i.p. with 10' allogenic spleen cells from the appropriate strains. Tumour-bearing mice were produced by intramuscular (i.m.) injection of 106 tumour cells into one hind leg. Cytostasis assay. Lymph nodes were removed from immunized mice between 10 to 15 days after immunization and single cell suspensions were prepared in a mixture of RPMI 1640 and MEM (Wellcome) supplemented with 5% foetal calf serum (Gibco). Tumour targets were obtained from the ascites of the appropriate mouse strain, washed twice and suspended in the above medium. Allogeneically or syngeneically immunized effector cells were mixed with tumour targets at ratios of 5:1 and 20:1, respectively, in a total volume of 1-0 ml in Greyward 1158 culture tubes. Lymphocytes from normal mice were mixed at the same ratios with tumour targets for control purposes. The tubes were incubated at 37°C for 48 hr in a 5% CO2 atmosphere. During the last 16 hr they were pulsed with [14C]thymidine. The cells were harvested on fibre filter strips using a semi-automatic harvesting machine and the isotope uptake was determined using a liquid scintillation spectrometer. In preliminary experiments it was shown that the uptake of isotope by the immune (IL) or normal (NL) lymphocytes was negligible during this period of incubation (Chia & Festenstein, 1973), thus the counts were taken to represent the proliferation of tumour cells. Cytostasis of tumour cells was expressed as a percentage inhibition of growth calculated according to the following formula (Senik et al., 1974a): percentage inhibition = 1000
tumour cells+IL -t/min ofof normal cells+NL
ct/min
Metabolic inhibition (cytostasis) assay using macrophage targets. This was carried out as previously described (MatossianRogers & Festenstein, 1977). Briefly, 5 x 104 peritoneal macrophages in culture medium were dispensed in each well of flat-bottom Microtest II plates (Cooke N/220-29AR, Cooke Laboratory Products, Alexandria, Virginia), incubated for a minimum of 2 hr at 37°C in a 5% CO2 atmosphere and washed to remove non-adherent cells just before use. TABLE 1. Tumour target cells: induction and strains of origin
Tumour cells
MBL2 (leukaemia) Gil4 (lymphoma) SL2 (lymphoma) LSTRA (sarcoma) P815-y (mastocytoma) Meth.A (lymphosarcoma) Gardner (lymphosarcoma) YAC (lymphoma) 4RLM (leukaemia)* *
Induction
Moloney virus Graffi virus Spontaneous Moloney virus
Methylcholanthrene Methylcholanthrene Oestrogen Moloney virus Moloney virus
Strains of
H-2
origin
haplotype
C57BI/6 C57BI/6 DBA/2 BALB/c DBA/2 BALB/c CBA/H A/Jax B1OA. (4R)
The 4RLM tumour was induced in our laboratory by Dr Giovanni Biasi.
H-2b H-2b
H-2d H-2d H-2d H-2d H-2k H_2a H-2h4
Cytostasis against self-antigens
211
Equal volumes of responder and irradiated stimulator lymphocytes at 2-5 x 106 per ml were cultured for 4 days; effector cells were removed and overlayed on macrophage targets at a ratio of 5 1. Normal cells were used at the same ratio for control purposes. The plates were incubated for 48 hr at 37'C in a 5%Y CO2 atmosphere. The effector cells were then washed off and 200 pl of culture medium together with 10pl of 14Clabelled uridine (0-33 pCi/mIl) were added to each well. The uptake of isotope during a further 18 hr incubation period was measured. The inhibition of isotope uptake was calculated using the formula: ct/min of macrophages + IL 1 100. percentage inhibition = 100- crp ct/min of macrophages+NLj Experimental design. Our previous experiments, using alloimmune effector cells across H-2 region incompatibilities demonstrated that cytostasis against H-2 coded alloantigens on tumour cells could be achieved if H-2K or H-2D region differences were presented together with an I-B region incompatibility during the in vivo sensitization. In the present experiments, we first showed that cytostasis occurred against syngeneic targets using effector cells from mice immunized with I-B region incompatible cells. To examine the targets of this cytostasis, combinations of B10 congenic mice and selected F1 hybrids were used to obtain incompatibilities in the I region alone. These combinations with I region differences only are henceforth termed 'syngeneic'. Cytostasis was achieved against tumour targets sharing the K- or D-end antigens of the responder cells. To confirm that cytostasis was directed against self-antigens, macrophages syngeneic to the responder lymphocytes were also used as targets in a post-labelling assay measuring 14C-labelled uridine uptake. This assay detected diminished metabolic activity of the target cells after treatment with immune cells as compared to normal cells, and is probably mediated by mechanisms similar to those that effect the stasis of tumour cell proliferation. Further experiments examined the specificity of cytostasis in immunizations with syngeneic tumour cells.
RESULTS A11oimmunization can induce 'syngeneic' cytostasis Specific cytostasis against self-antigens expressed by K-end genes of the H-2 complex is obtained by immunizing mice with an effective I-B region incompatibility only. Table 2 shows several combinations where immunization across this restricted incompatibility is achieved by the use of F1 hybrids as responders. Effector cells obtained from [B1O.A(4R) x BIO.A(5R)]F1 mice immunized against B1O.A (I-B region incompatibility) were cytostatic for tumour cells bearing syngeneic K-end specificities (Gardner) but not syngeneic D-end specificities (MBL2). This represents 'syngeneic' cytostasis, since the responder strain also expressed H-2k antigens at the K end. Two further combinations [B1O.A(4R) x B1O.D2]F1 immunized against B1O.A(2R) and [B1O.A(4R) x B1O.D2]F1 immunized against B1O.A yielded cytostatic effector cells for tumours expressing syngeneic K-end specificities. It is unlikely that the cytostasis in these combinations is directed against the I-B region specificities. B1O.A(4R) anti-B1O.A(2R) effector cells, which represent b anti-k at the I-B region, were cytostatic for tumour cells carrying both K-end (H-2k, Gardner) and D-end (H-2b, Gil 4) specificities of the effector cells. The D end in this combination bears no apparent relationship to the specificity of the I-B region immunization. An analogous situation was noted with B1O.A(2R) anti-B1O.A(4R) effector cells, where cytostasis of the H-2k tumour (Gardner) did not follow the specificity of the I-B region incompatibility, which was k anti-b (Table 2). If the incompatibility between BlO.A(4R) and B1O.A(2R) was narrowed down to the I-B region only by immunizing a [B1O.A(4R) x B1O.D2]F1 hybrid against B1O.A(2R), then cytostasis was directed only against tumours sharing K-end antigens of the effector cells. It can be concluded from these experiments that an I-B region incompatibility generates cytostasis against K-end syngeneic targets, irrelevant of the specificity of the I-B region immunization, and additional I region incompatibility is necessary to obtain cytostasis to D-end targets. Cytostasis using macrophage targets The cytostatic potential of effector cells immunized against I region incompatibilities alone was tested against macrophage targets in a metabolic inhibition assay. B1O.A(2R) anti-B1O.A(4R) effector cells were strongly cytostatic for B1O.A and B10 macrophages
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which share K-end and D-end specificities, respectively, of the responder strain, and also for syngeneic macrophages and B10.A(4R) macrophages, which are identical at the K end and D end with the responder lymphocytes (Table 3). When B1R.A was immunized against B1O.A(4R), providing I region and D-end incompatibilities, cytostasis was directed against the H-2b alloantigen (B10 macrophages) and also against self-antigens expressed on syngeneic (B1O.A) and allogeneic macrophages [B1O.A(4R) and B1O.A(2R)]. These results demonstrate that the targets for cytostasis in combinations incompatible for I region alone are selfantigens.
Cytostasis against syngeneic tumours Immunization of pure inbred or recombinant mice against syngeneic tumours resulted in the generation of cytostatic effector cells against syngeneic tumour targets of the pure haplotype or K-end and D-end specificities of the recombinant haplotypes. Effector cells from B1O.A(4R) mice immunized with syngeneic tumour cells (4RLM) (Table 4) were cytostatic for YAC and Gardner tumours syngeneic with the effector cells at the K end and also for MBL2 syngeneic at the D end. BALB/c mice immunized against the syngeneic Meth.A tumour were cytostatic only for H-2d-bearing targets, such as YAC and Meth.A, and not for H-2b (MBL2) or H-2k (Gardner). Immunization of A/Jax (H-2a) and B10.A (H-2a) mice with YAC tumour syngeneic to A/Jax resulted in an interesting difference. A/Jax effector cells were cytostatic to both K-end and D-end targets, while B10.A effector cells were cytostatic to K-end targets only. The restricted effector capacity in the latter case may be explained in the light of the previous data in that YAC tumour cells may have presented a narrower range of incompatibility to B10.A responder lymphocytes than to A/Jax lymphocytes. This will be further discussed. DISCUSSION Virally or chemically induced tumours carry transplantation antigens which stimulate the development of effector cells in syngeneic hosts. The nature of these transplantation antigens is still unclear; however, they induce or become targets for cytotoxic cells (Brunner & Cerottini, 1971) or, as we have demonstrated in this report (Table 4), for cytostatic effector cells. These two immune phenomena are distinct in their mechanism and even though they may coexist in vivo, they are separable in vitro (Plata et al., 1974; Seeger & Owen, 1973). We have previously shown that to generate cytostatic effector cells directed against MHC gene products, an I-B region incompatibility between responder and stimulator lymphocytes is essential (De Giorgi et at., 1977). Cytotoxic cells, however, can be produced in I-B region compatible combinations of recombinant C57B1/10 congenic mice (Matossian-Rogers, Demant & Festenstein, 1975). This is an important difference in the sensitization phase of the two effector systems. If I-B region incompatibility is presented together withH-2K or H-2D region differences, thencytostasis against these alloantigens on tumour targets is achieved. If I-B region incompatibility is presented alone, cytostasis still occurs and is directed against syngeneic K-end antigens only, while a broadening of the I region incompatibility extends the specificity of syngeneic cytostasis to D-end targets as well. Brondz, Egorov & Drizlikh (1975) showed that different clones of T cells are specific for H-2K or H-2D antigens. It is possible that clones of lymphocytes with helper or effector function with restricted specificity may be differentially activated by various regions of the H-2 complex. To explain the generation of cytostasis against syngeneic tumour targets on the basis of the incompatibility presented by TSTA, we postulate that the viral or chemical induction of tumours may be associated with an I region modification of the neoplastic tissue. The extent of this modification then determines whether cytostasis will be directed to K-region or D-region antigens. The differential response of A/Jax and B10.A may be explained by this hypothesis, if we assume that they have minor I region differences (hitherto undetected) and that the modified I region of the YAC
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216
Luisa De Giorgi, Arpi Matossian-Rogers & Hilliard Festenstein
tumour is more similar to the I region of the B1O.A mouse. As a corollary, these results would then be the first indication of I region differences in A/Jax and B1O.A mice! The nature of the I region modification which initiates an immune response to self-antigens is not clear. Though it may be a serologically detectable new Ia specificity, and Ia specificities of foreign haplotypes have been found on SL2 tumour cells (Garrido, Festenstein & Schirrmacher, 1976; Hammerling et al., 1974), no known Ia specificity has yet been definitively mapped in the I-B region (Shreffler, Meo & David, 1977), suggesting that it is more likely to give rise to a cell-mediated response. Evidence that tumour-specific transplantation antigens are modified histocompatibility antigens has been previously demonstrated by Bowen & Baldwin (1975). The question still remains how a modification in the I region breaks the tolerance to self-antigens and makes them targets for immune effector cells. Since cytostasis is directed against K- or D-region antigens on autochthonous and other syngeneic tumours (Table 4), the specificity of these gene products does not appear to be altered by the oncogenic agent. Nevertheless, allosteric alteration, possibly by mechanical distortion, may take place without affecting specificity, thus providing a new acceptor site for recognition structures on immunocompetent cells. This may be analogous to some interactions of virus-immune lymphocytes against syngeneic virustransformed target cells for viruses that may not cause any serologically detectable modification of the cell membrane (Trinchieri, Aden & Knowles, 1976). On the other hand, no alteration in structure may be necessary if a two-receptor hypothesis is invoked for self-recognition (Doherty et al., 1976). The V-region genes may be phenotypically expressed on the lymphocyte membrane, one recognizing H-2K or H-2D and the other possibly an I region modification. Recognition by one signal alone, for example the idiotype for H-2K, may not provide sufficient avidity for the effector phase, while multiple recognition by two or more idiotypic receptors would achieve the threshold for immunological function. Such a system would be compatible with any mechanism that may effect cytostasis, since both T and B lymphocytes directed against the same alloantigens have been shown to use receptors with shared idiotypic determinants (Binz & Wigzell, 1975a,b). Finally, our results may shed some light upon self-recognition mechanisms in autoimmune phenoma. Genes mapping at H-2K or H-2D function as self-markers in cell-mediated immunity, as shown in systems using virus-infected syngeneic cells. The classical HLA antigens in man may have similar functions, while modification of antigens analogous to those encoded by the I region, possibly the B lymphocyte antigens, may be the triggering mechanism for autoimmune damage. We thank Dr Giovanni Biasi for inducing the 4RLM tumour and the Cancer Research Campaign for financial support.
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