Nonspecific activation of murine spleen cells in vitro by a synthetic immunoadjuvant (N-acetyl-muramyl-L-alanyl-D-isoglutamine)

We reported previously that synthetic N-acetyl-muramyl-l-alanyl-d-isoglutamine (MDP) displayed marked adjuvant activity but was devoid of mitogenicity in vitro. The data reported here establish that, under different cultural conditions, thymidine uptake and blast cells can be increased by MDP in spleen cells of DBA/2 and Balb/c mouse strains. Optimal responses were obtained on culture in a serum-free medium supplemented with 2-mercaptoethanol for 4 or 5 days. This effect was also obtained with spleen cells of Balb/c nude mice. When the synthetic MDP was compared to a natural water-soluble adjuvant (neo-WSA), extracted from Mycobacterium smegmatis cells, both were found to stimulate [³H] thymidine incorporation by mouse spleen cells. However, with the neo-WSA, the effect peaked on Day 2 and was weak or absent on Days 4 and 5. When the cells were cultured in a medium containing fetal calf serum, neo-WSA activation was completely abolished, while MDP-mediated stimulation was decreased.     

Variations in the response of five strains of mice to Leishmania mexicana.

Five strains of mice were studied in their ability to support Leishmania mexicana infection. Four strains, AKR, C57BL/6, DBA/2 and NMRI, were relatively resistant to cutaneous leishmaniasis. These strains developed delayed type hypersensitivity responses to leishmanial antigens and produced agglutinating antibodies. On the other hand Balb/c mice, highly susceptible to infection, failed to develop delayed type hypersensitivity responses and showed an impaired production of antibodies. Hybrids produced by mating C57BL/6 males and Balb/c females were no more susceptible than C57BL/6 mice, suggesting that resistance is inherited as a dominant character.     

In vitro production of immune interferon by spleen cells of mice immunized with herpes simplex virus.

In Vitro production of Immune Interferon (IF) in response to Herpes Simplex Virus (HSV) antigen by sensitized spleen cells from C57B1/6 (B6) mice could be detected as early as 3 and for at least 20 days after ip infection of HSV. Maximal levels of IF were produced after 10 hr of culture, but there was no decay of activity when supernatants were sampled during the subsequent 3 days. The IF produced shared certain known properties of immune IF and was not neutralized by an antiserum against viral-induced (type I) IF. DBA/2 (D2) mice which are considerably more sensitive in vivo to HSV infection than B6 mice produced significantly lower amounts of immune IF in the in vitro test system regardless whether high or low doses of virus were injected. The same pattern of results was observed when resistant B6D2F1 hybrid mice were compared with $\text{A}\text{J}$ and Balb/c mice which are about as sensible to ip infection with HSV as DBA/2 mice in our laboratory. These results demonstrate a remarkable defect of in vitro cellular immunity in mice susceptible to a virus infection when compared with resistant mice. Conceivably, a similar defect may be of in vivo relevance.     

Soluble factors in mouse immune sera: III. Origin,nature, and target of small molecular weight suppressive factors isolated from serum during primary responses

Small molecular weight suppressive factor (s) (< 10,000 daltons) were separated by Diaflo filtration from sera of Balb/c mice undergoing a primary response to sheep erythrocytes. These factors could be induced only when both T and B cells were challenged with antigen simultaneously. Thymus or bone marrow cells exposed in vitro to these factors showed marked impairment of their ability to collaborate for antibody synthesis in adoptive transfer experiments. These data suggest that both the T and B cells form the target for these factors. When the fractions showing suppressive activity were examined over a wide dose range, no enhancing activity was detected. The suppressive factors lost their activity after treatment with Pronase or heating at 63 °C for 30 min, but they were resistant to digestion with RNase.When a variety of mouse strains was examined for the production of small molecular weight suppressive factors it was found that certain strains produced factors which suppressed both 19 and 7S responses (Balb/cJ, AKR, and SJL/J), while in others factors affecting only the 7S response (A/J and C3H/He) or only the 19S response CBA/J) were detected. Finally, in some strains no suppressive activity was recovered (DBA/2 and B10D2), while in C57BL/6J distinct enhancing activity was detected. The F₁ hybrids of C57BL × Balb/c produced neither suppressive nor enhancing activity.     

Plaque-forming cells fater in vitro stimulation with theta-AKR antigen

Spleen cells of inbred mice strains carrying θ-C3H allele have been cultured in the presence of AKR thymus cells and their in vitro primary PFC response against thymic alloantigen θ-AKR was studied.The responses of a magnitude which was comparable with that obtained in previous in vivo experiments were obtained 4 days after stimulation. The strain-dependent variability of the magitude of anti-θ-AKR responses was observed in vitro. RR and C58/J spleen cells produced much more PFC than C57BL/6J and DBA/2J spleen cells. This was in agreement with previous in vivo studies on the genetic control of the anti-θ AKR responses.In the absence of AKR thymus cells, spleen cells of high responders, RR, developed in vitro PFC which released antibodies lytic to AKR thymus cells. Their number was ten-times lower than in stimulated cultures. Spleen cells of all strains tested produced also small numbers of PFC secreting antibodies against θ-identical allogenic thymus cells and even to syngenic thymus cells.     

Transplantation antigen expression on murine trophoblast detection by induction of specific alloimmunity

Cell-mediated immune responses to murine embryonic trophoblast cells were investigated using lymphocyte trophoblast cultures (LTC) and cell-mediated lympholysis (CML). Spleen cells from CBA (H-2^k) or C57BL/6 (H-2^b) mice hyperimmunized with 3.5-day-old Balb/c (H-2^d) blastocysts did not undergo DNA synthesis after in vitro exposure to Balb/c blastocyst outgrowths nor were cytotoxic lymphocytes (CTL) generated against H-2^d alloantigens. Splenocytes from Balb/c mice presensitized with semiallogeneic (Balb/c female × C57BL/6 male) trophoblast cells derived from 17- to 20-day placental tissue expressed a weak proliferative response in the presence of semiallogeneic placental trophoblast and produced a moderate number of CTL against H-2^b (paternal strain) alloantigens when compared to mixed lymphocyte cultures (MLC) between Balb/c responder and semiallogeneic (stimulator) spleen cells. CTL were also generated in vitro after splenocytes from Balb/c mice hyperimmunized with semiallogeneic spleen cells were restimulated in vitro with placental trophoblast cells. These studies showing that early-stage trophoblast cells fail to evoke transplantation immunity and placental trophoblast is capable of generating alloimmunity only after combined in vivo hyperimmunization with in vitro restimulation suggest that these trophoblast cells are poorly immunogenic due in part to the relatively weak functional expression of major transplantation antigens.     

Leishmania tropica: pathogenicity and in vitro macrophage function in strains of inbred mice.

Of seven strains of inbred mice and one hybrid that were infected intracutaneously with 5, 10, or 20 × 10⁶ active promastigotes of Leishmania tropica major, two strains (CBA/Ca and C₃H/He) recovered from the infection and their lesions healed within 3 to 5 months. The other strains, with the possible exception of C57B1/6 animals, remained infected, carrying large cutaneous ulcers throughout their lives. These included DBA/2, A/Jax, Balb/c, athymic nude mice of Balb/c origin (nu/nu) and the heterozygote Balb/c (nu⁺). The responses of C57B1/6 animals were of intermediate type with a tendency toward nonhealing at higher doses of the parasite. The cutaneous infection of athymic nude mice invariably gave rise to fulminating visceral infections and death. This condition was never observed in the other strains tested. Concanavalin A (Con A)-stimulated syngeneic or allogeneic lymphocytes of intact mice activated peritoneal macrophages of both healer and nonhealer mice, resulting in complete destruction of phagocytosed L. enriettii within 24 to 48 hr. The destruction of ingested L. tropica was confined to macrophages of healer mice and required 72 to 96 hr to reach completion. However, removal of Con A-stimulated lymphocytes from macrophage cultures and regular pulsing of the cells with a lymphokine-rich supernatant produced a state of sustained activation, resulting in destruction of L. tropica inside macrophages of both healer and nonhealer mice. The ability of Con A-stimulated lymphocytes of nonhealer animals to induce effective levels of activation in healer macrophages on one hand, and eventual destruction of L. tropica in macrophages of nonhealer mice under condition of sustained activation on the other, had indicated that so far as the in vitro situation is concerned, there is no inherent defect in lymphocytes or macrophages of nonhealer animals, although the threshold of activation necessary for killing of the parasite seems to be higher for cells of nonhealer origin.     

Polyclonal B Cell Activation by Cell Wall Preparations of Gram-Positive Bacteria

The effects of polyclonal B cell activation (PBA) of cell walls and their cell wall fractions obtained from several kinds of gram-positive bacteria were studied using the anti-sheep red blood cell (SRBC) or anti-trinitrophenylated (TNP) SRBC plaque forming cell (PFC) responses of cultured spleen cells from Balb/c, athymic nu/nu, their littermates (nu/+), C3H/He (LPS-responder), C3H/HeJ (LPS-non-responder), (CBA/N × Balb/c) F1 male with an X-linked defect in B cell function and the F1 female mice. The cell walls of Staphylococcus epidermidis (ATCC 155), Lactobacillus plantarum (ATCC 8014), Micrococcus lysodeikticus (NCTC 2665), Mycobacterium rhodochrous (ATCC 184), Streptomyces gardneri (ATCC 23911) and Nocardia corynebacteriodes (ATCC 14898) had the ability to induce polyclonal B cell responses in the spleen cells of Balb/c, nu/nu, nu/+, C3H/He and C3H/HeJ mice. The cell wall fractions prepared by enzymatic digestion from the cell walls of S. epidermidis, S. gardneri or N. corynebacteriodes were also capable of inducing polyclonal B cell responses. The responses of spleen cells from (CBA/N × Balb/c) F1 male mice to these active preparations, except the cell walls of M. rhodochrous, were much lower than those of the F1 female mice. These findings indicate that the majority of the cell wall preparations lacks PBA ability for spleen cells with the CBA/N defect, except for the cell walls of M. rhodochrous which possess this ability. The PBA-ability of synthetic peptidoglycan, muramyl dipeptide (N-acetylmuramyl-L -alanyl-D -isoglutamine, MDP), was also examined, and a similar activity was observed in MDP.     

Relationship between induction of aryl hydrocarbon hydroxylase and de novo synthesis of cytochrome P-448 (P1-450) in mice

Phenobarbital, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), benzpyrene, 3-methylcholanthrene (3-MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were administered i.p. for 1 or 3 days to genetically “responsive” (C57BL/6J) and genetically “non-responsive” (DBA/2J) mice. 3-MC or benzpyrene stimulated aryl hydrocarbon hydroxylase (AHH) activity in C57BL/6J (B6) mice but not in DBA/2J (D2) mice. TCDD induced AHH activity in both B6 and D2 mice. Time-course studies showed that in the first 12 h after a single injection of 3-MC to B6 mice there was no shift in the reduced cytochrome P-450-CO complex absorption spectra from 450 to 448 nm, although AHH activity increased 4–5 times over (above) that of the control group. The relationship between induction of AHH activity by polycyclic hydrocarbons in B6 mice and the concomitant synthesis of cytochrome P-448 is discussed.     

Immunogenetic differences between lymph node cell and bone marrow cell grafts in irradiated mice

C3H lymph node cell (LNC) grafts, but not bone marrow cell (BMC) grafts, were resisted by lethally irradiated NZB, (C57BL × NZB)F1, and (C57BL/6 × DBA/2)F1 mice. BALB/ c hosts did not resist C3H LNC, suggesting that Ir-like genes regulate resistance to such grafts. Cyclophosphamide, silica particles, and ⁸⁹Sr pretreatments of prospective host mice resulted in successful proliferation of C3H LNC in most instances. These agents were known to abrogate resistance to incompatible BMC grafts. The determinants for antigens recognized on LNC appear to map in or near the D region of H-2. LNC grafts of all H-2^k strains tested (C3H, CBA, C58, C57BR) were strongly resisted while A, C3H.A, B10.A(5R), A.TL, and A.Tla^b LNC grafts were not strongly resisted by NZB hosts. Grafts of H-2^b (C57BL/6, C57BL/10, 129) LNC, or BMC are resisted by NZB or (C57BL/6 × DBA/2)F1 hosts. (C3H × C57BL)F1 LNC but not BMC were resisted by similar hosts. (C57BL/6 × DBA/2)F1 mice were injected with C57BL/6 spleen cells four times to induce specific “unresponsiveness” to parental-strain Hemopoietic histocompatibility (Hh) antigens. Unresponsiveness was induced to C57BL/6 BMC, as expected, but C57BL/6 and C3H LNC grafts were resisted despite the spleen cell injections. The data suggest that the antigens recognized during rejection of C3H LNC are not expressed on C3H BMC. It is even conceivable that Hh antigens on C57BL/6 BMC and LNC have separate determinants. Alternatively, the injections of C57BL/6 spleen cells may have induced an anti-idiotypic response that was capable of eliminating C57BL/6 LNC by a different effector mechanism.     

Augmentation of mouse natural killer cell activity by muramyl dipeptide and its analogs

The ability of muramyl dipeptide (MDP) and its structural analogs (des-MDP, abu-MDP, and des-abu-MDP) to influence mouse natural killer (NK) cells in two different strains of mice was examined. In CBA/J mice, administration of MDP by both intraperitoneal (ip) and intravenous (iv) routes enhanced splenic NK cell activity. Maximum augmentation of NK cell activity was observed 3 days after MDP treatment. NK cell activity was also stimulated upon in vitro culture of CBA/J mouse spleen cells with MDP. Only iv inoculation of MDP to C57BL/6 mice 7 days previously enhanced NK cell activity of spleen cells. Peritoneal NK cell activity was not affected in either strain of mice, regardless of the route of inoculation of MDP. Two structural analogs of MDP, abu-MDP and des-abu-MDP, enhanced peritoneal NK cell activity, whereas des-MDP had no effect when tested 3 days after ip treatment of CBA/J mice with these compounds. Peritoneal NK cell activity of C57BL/6 mice was not modulated by des-MDP, abu-MDP, or des-abu-MDP. A synergistic effect on peritoneal NK cell activity was observed in both CBA/J and C57BL/6 mice treated first with MDP and then with lipopolysaccharide (LPS) or Bacillus Calmette-Guerin (BCG).     

Inhibitory and stimulatory effects of a synthetic glycopeptide (MDP) on the in vitro PFC response: factors affecting the response.

A synthetic adjuvant active glycopeptide, N-acetyl-muramyl-l-alanyl-d-isoglutamine (MDP), has been previously shown to enhance the in vitro immune response of mouse spleen cells to T-dependent or independent antigens. Data presented here show that the net activity of MDP on the in vitro immune response is closely related to the cell culture conditions: Two distinct patterns of MDP activities could actually be detected. Marked stimulation of the PFC response was observed at “low density” cell cultures. In contrast, suppression could be seen at “high density” cell cultures. Moreover, the culture conditions which permitted characterization of either the enhancement or suppression of the immune response by MDP were strongly dependent on the strain of mouse used. However, these activities were not dependent on antigen concentration, on kinetics of responses, or on cytotoxic effects.     

Varying susceptibility of pulmonary cytokine production to lipopolysaccharide in mice

Objectives: The lipopolysaccharide (LPS)-induced acute lung injury (ALI) model has been widely applied for pathophysiological and pharmacological research. The aim of present study is to understand the variation of acute pulmonary inflammation between mouse strains. Methods: The present study investigated the susceptibility of acute production of inflammatory mediators, e.g. cytokines, chemokines and others, to LPS in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J, and C3H/HeN mice. Results: The susceptibility to intra-tracheal challenge with LPS varied between measured variables, durations and strains. General lung hyper-reactive susceptibility to LPS-induced pulmonary production of 6–8 inflammatory mediators followed the order NMRI, Balb/cJ, C3H/HeN, A/J, C57BL/6J, DBA/1J, DBA/2J and CD-1 mice at 4 h, and A/J, C3H/HeN, CD-1, NMRI, C57BL/6J, Balb/cJ, DBA/2J and DBA/1J mice at 24 h. Conclusions: Our data provide information for scientists to consider the proper strain of mice for the measurement of specific inflammatory mediators and to select sensitive or resistant mouse strains for understanding genetic variation in the pathogenesis and for the screening of target-oriented drug development.     

The adjuvant activity of synthetic N-acetylmuramyl-dipeptide: evidence of initial target cells for the adjuvant activity.

MurNAc-l-Ala-d-isoGln (N-acetylmuramyl-l-alanyl-d-isoglutamine, MDP), a synthetic compound, acts as an adjuvant on the humoral immune response and on the T cell-mediated immune response. In this report, we attempted to directly demonstrate the initial target cells of MDP for its adjuvant activity in vitro by using cell separation procedures.It was demonstrated that MDP enhanced the immune response following direct interaction with antigen-stimulated T and B lymphocytes, but nonstimulated lymphocytes, shortly after triggering by antigen, and that there was no macrophage requirement for MDP to elicite the adjuvant action in the primary anti-SRBC PFC response in vitro. It has also been demonstrated that the adjuvant activity of MDP is due to an enhancing effect which is different from the possible mitogenic activity to spleen cells and MDP replaces neither a function of macrophages, which is substituted by 2-mercaptoethanol nor a helper function of T cells.     

Suppressor factor from tumor-allosensitized spleen cells--its effect on in vitro proliferation of tumor cells and in vivo skin allograft survival

C57BL/6 mice are sensitized ip with allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the sensitized mice are used in the production of suppressor factor or treated with mitomycin and used as suppressor cells. Sensitized spleen cells incubated with the specific alloantigen (DBA/2 m-treated spleen cells) release suppressor factor (SF)² which inhibits cell proliferation in mixed lymphocyte culture (MLC) as well as the in vitro generation of cytotoxic cells (CML). SF is most effective when added eary during MLC. SF also inhibits mitogen responsiveness of normal spleen cells. In addition to inhibiting lymphocyte function in vitro, suppressor cells as well as SF inhibit the in vitro proliferation of tumor cells. This inhibition is specific for the tumor to which the suppressor cells are induced. The inhibition of tumor cell proliferation is not due to the presence of cytotoxic cells in the spleen of the tumor-allosensitized mice. Suppressor cells from neonatal mice do not inhibit the in vitro proliferation of tumor cells. SF injected iv into C57BL/6 mice decreases the mixed lymphocyte reactivity of the host spleen cells and decreases the ability of the host to reject skin allografts. We interpret these data to suggest that tumor-allosensitized spleen cells, and the SF they produce, not only affect lymphocyte function but also inhibit tumor cell proliferation. This dual effect of suppressor cells could be an important part of the immune surveillance against tumors.     

The mixed lymphocyte response of senescent mice: sensitivity to alloantigen and cell replication time.

The age-dependent alteration in the proliferative response of C57B1/6J lymph node cells to stimulation by H-2- and M-locus alloantigens was examined in one-way mixed lymphocyte cultures (MLC). Balb/c (H-2^d, Mls^b) and DBA (H-2^d, Mls^a) spleen cells served as stimulating cells differing from C57B1/6J (H-2^b, Mls^b) at the H-2 and H-2 plus Mls loci, respectively. The day of peak response and the ratio of responder to stimulator cells required for optimal stimulation were the same for all the age groups (3 to 29 months) tested, irrespective of the stimulator strain used. Results obtained in MLC under optimal conditions showed a maximal response to both Balb/c and DBA/2 stimulation at the age of 6 months, followed by a gradual decline in the response with age. In order to determine whether the decline with age in mixed lymphocyte reactivity can be attributed to a reduction in the proliferative capacity of the responding lymphocytes of aged mice, cell cycle analyses were performed. Auto-radiographic studies of MLC containing lymphocytes from CS7B1/6J mice aged 6 and 24 months showed no difference in generation time, S, G₂, G₁, and M phases of the cell cycle. In addition, lymphocytes of both age groups underwent two identical mitotic waves within the period of examination. Our results determine that the functional decline with age in proliferative activity in mixed lymphocyte cultures is attributable to a neither decrease in sensitivity to alloantigen nor to a decrease in generation time or the ability to undergo several mitotic divisions, and suggest that such a decline is caused by fewer cells capable of response in old mice.     

Enhancement of carrier-specific helper T cell function by the synthetic adjuvant, N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP).

The adjuvant effect of a synthetic peptidoglycan, muramyl dipeptide (N-acetyl muramyl-L-alanyl-D-isoglutamine, MDP), was studied by using the anti-Tnp PFC and hemagglutinin responses of BALB/c mice to hapten-carrier conjugates. Administration of Tnp-OVA and MDP in saline to mice, followed 2 weeks later by a boost of Tnp-OVA in saline, led to significantly higher IgM and IgG anti-Tnp PFC and total anti-Tnp-hemagglutinin responses than those obtained in mice not treated with MDP in the initial immunization. A similar adjuvant effect by MDP on anti-hapten PFC responses was seen if mice were primed with KLH together with MDP and challenged with Tnp-KLH 2 weeks later. This apparent effect on carrier priming for helper function was confirmed and quantitated by double adoptive transfer experiments with graded numbers of spleen cells from KLH +/- MDP-primed mice and a fixed number of hapten-primed spleen cells from syngeneic Tnp-OVA immunized animals. These data suggest that at least one mode of action of the synthetic adjuvant MDP is via the enhanced stimulation of the helper T cell function.     

Specific antigens inhibit plasmacytoma cells bearing phosphorylcholine-binding myeloma proteins.

The influence of a synthetic adjuvant active glycopeptide, N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP), and of some of its analogs on the in vitro immune response to sheep red blood cells was studied using Mishell and Dutton in vitro stimulation system. When MDP and adjuvant active analogs were incubated with normal spleen cells, increased cell recovery was observed after 3 or 4 days of culture, showing a good correlation between the adjuvant activity in vivo and the enhancement of cell viability in vitro. The analogs which were found to have an adjuvant activity in vivo were equally effective in stimulating in vitro both the background hemolytic PFC and the immune response to sheep red blood cells. However, those which were inactive in vivo were effective in vitro but only at high concentration levels.     

In vitro sensitization to transplantation antigens. VII. Enhanced graft-vs-host activity of in vitro allosensitized lymphoid cells

We have previously demonstrated that C57BL/6J lymphoid cells sensitized in vitro to C3H/He transplantation antigens, present on macrophage monolayers, can transfer an accelerated C3H allograft response to recipient C57 mice. The present report indicates that C57 lymphoid cells sensitized to C3H alloantigens, present on macrophage monolayers, can also mediate a graft-versus-host (GVH) reaction in (C3H × C57) F₁ newborn mice. This GVH reaction is of greater magnitude than that produced by noncultured C57 cells. The magnitude of the augmented GVH reaction produced by cultured C57 cells is dependent on the source of lymphoid cells: lymph node, spleen, and bone marrow cells are consistently more active than cultured thymus cells—the reduced capability of cultured thymus cells to mediate the GVH reaction parallels their reduced ability to transfer allograft immunity. To test whether monolayers, other than macrophages, can sensitize lymphoid cells in vitro we incubated C57 lymphoid cells on C3H-derived L cells. Lymph node cells incubated with L cells demonstrate an increased GVH reaction in newborn mice. The in vitro sensitization of spleen and bone marrow cells on L cells is less consistent. Thymus cannot be sensitized by L cells. Monolayers of L cells are therefore not as efficient a sensitizing source as macrophages.     

The effect of anti-lymphocyte and anti-thymocyte serum on the pathogenesis of Rauscher leukaemia in inbred mouse strains.

Rauscher virus causes in inbred Balb/c mice a rapidly progressing hyperacute, in C57B1/10Sn mice an incipient, spontaneously healing leukaemia. In DBA/1 and DBA/2 mice the appearance of leukaemia is followed by a partial remissions then by an exacerbation of the disease. The infection in C57B1/10Sn, DBA/1 and DBA/2 mice results in a significant tumour-specific immune response. Inhibition of the immune response is followed by an increased progression of leukaemia in DBA/1 and DBA/2 mice only. It is assumed that in C57B1/10Sn mice the remission of incipient leukaemia is associated with a resistance determined in the target cells, whereas in DBA/1 and DBA/2 mice the remission is due to a tumour-specific immune response. As in animals treated with anti-lymphocyte and anti-thymocyte serum the course of the disease runs proportionally to the degree of the inhibition of the immune response, the tumour-specific antibodies play a decisive role in the elimination of the tumour cells. In Balb/c, DBA/1 and DBA/2 mouse strains failing to exhibit a spontaneous reversion of the tumour cells, the appearance of a significant tumour-specific immune response depends on the resistance against the helper component of the Rauscher virus complex.