Immunotherapy II: Antigens, receptors and costimulation

To generate a cytotoxic T-lymphocyte (CTL) response to cancer cells requires tumour-specific antigens appropriately processed and displayed by the MHC proteins; T-lymphocytes with receptors of appropriate specificity to recognise these; and initial antigen presentation to the immune system in an immunogenic context. In vitro, autologous tumour-specific CTL have been raised against a number of tumours, thus at least some patients have a suitable combination of antigen and receptor. Vaccination with antigen, or with DNA or viral vectors encoding the antigen, leading to the presentation of identified antigens in an immunogenic context, can activate T-cells which provide protection from tumour in animal models. An alternative approach uses gene transfer to T-cells, causing them to express novel receptors which direct their cytotoxic activity towards the tumour. Non-specific immune adjuvants, and expression of novel antigens on tumour cells, are briefly discussed. Recent advances in understanding the requirements for T-cell activation suggest that failure to efficiently present antigen in an immunogenic context may explain the apparent lack of tumour-specific CTL activation in vivo. In mice, expression of the costimulatory molecule B7-1 on tumour cells, following gene transfer, allows the modified tumour cells to act as antigen-presenting cells, inducing protective and therapeutic CTL responses in some cases. Clinical trials of some approaches have commenced, with some encouraging results which provide a basis for further development of immunological gene therapy.     

Immunity against moloney sarcoma virus in H-2Db mutant bm14 mice. Unimpaired tumor immunity despite absence of a virus-specific cytotoxic T-cell response

It was shown previously that B6.C-H-2^bm14 (bm14) mice, carrying a mutation in the H-2D^b locus, are unable to generate cytotoxic T cells (CTL) against Moloney murine sarcoma virus (M-MSV). We now report an analysis of tumor induction and regression kinetics and of immunity to the virus, following the injection of graded doses of M-MSV into C57BL/6 (B6) and bm14 mice. Contrary to expectation, bm14 mice showed slightly less tumors than wildtype B6 mice. Moreover, all bm14 mice that developed a tumor were able to reject this tumor, even after injection of the highest virus dose tested. From the spleen cells of bm14 mice that had rejected tumors, no secondary in vitro CTL responses could be generated, in contrast to strong CTL responses generated from B6 spleen cells. Although bm14 mice were unable to generate virus-specific CTL, they showed nomal antibody and T-cell proliferative responses against Moloney virus, suggesting an intact T-helper-cell function. It is conculded that in bm14 mice, under the conditions tested, virus-specific CTL are not generated despite excellent tumor immunity. Therefore, this CTL response is not necessary for protection against M-MSV-induced tumors. Protection is likely to be mediated by a normal T-cell proliferative response.     

Significance of cytotoxic lymphocytes after various immunizing procedures in a virus-induced non-producer syngeneic system: correlation between in vitro and in vivo lytic activity

An originally virus-induced, non-producer tumour system has been studied in relation to humoral and cellular cytotoxic responses to transplantation and other immunization techniques. In all experimental groups cytotoxic lymphocytes (CTL) were observed either directly or after mixed culture of lymphocytes and tumour cells (MLTC). Except for C'-dependent cytotoxic antibodies in mice immunized by irradiated cells, no antibody-mediated cytotoxicity was observed. In 2 protocols (transplantation and immunization by mitomycin-treated cells) CTL in vitro were not protective. In a third protocol (immunization by irradiated cells) CTL afforded partial protection and other factors appeared to be involved. The best in vivo protection was induced by immunization consequent on early surgical removal of a small number of transplanted tumour cells. This study provides lines of evidence for the effectiveness of protection supplied by CTL in well-defined conditions. Comparison with other modes of immunization indicated that these conditions were related to the quantity and to the characteristics of antigen involved.     

Immunotherapeutic potential of dendritic cells generated in long-term stroma-dependent cultures

Long term cultures (LTC) producing dendritic cells (DC) have been established from spleen. A well developed stromal cell layer supported production of DC in numbers suitable for experimentation. Cells had obvious membrane pseudopodia and could be collected from culture every 2-3 days. Large cells produced in LTC stained with fluorescently labelled monoclonal antibodies specific for DC such as 33D1, and M1/70 which is specific for DC and myeloid cells. These staining patterns confirmed the presence of DC within the LTC population. LTC-DC were tested and shown capable of migration in vivo in B10.A(2R) mice following footpad inoculation. Most cells entered the spleen and a small number entered popliteal lymph node. LTC-DC have migratory capacity comparable with control spleen lymphocytes. LTC-DC were tested for capacity to induce an anti-tumour immune response after exposing cells to tumour cell membranes. LTC-DC pulsed with BL/VL3 tumour antigens were able to induce a BL/VL3-specific primary cytotoxic T lymphocyte (CTL) response detectable in popliteal lymph nodes and spleen of C57BL/6J mice within 6 days of priming. BL/VL3 tumour cells grew in sublethally irradiated C57BL/6J mice giving 100% mortality. Adoptive transfer of spleen cells from mice given BL/VL3 antigen-pulsed LTC-DC, two weeks previously, significantly slowed the growth of BL/VL3 tumour cells in mice. DC produced in LTC can function as antigen presenting cells (APC) when adoptively transferred into animals. Their capacity to migrate effectively, to induce a CTL response and to reduce tumour load suggests that DC grown using this in vitro system may have valuable clinical potential in humans.     

Lysis of human pancreatic adenocarcinoma cells by autologous hla-class I-restricted cytolytic T-lymphocyte (CTL) clones

From the primary site of a pancreatic adenocarcinoma (patient BE) a permanent cell line (MZ-PC-2) was established in tissue culture. In the course of mixed lymphocyte-tumor-cell cultures (MLTC) with autologous blood-derived lymphocytes, we isolated CTL clones that lysed autologous tumor cells but not autologous EBV-transformed B cells (EBV-B) and not K562. Pre-treatment of MZ-PC-2 cells with IFN-γ was required to obtain significant lysis in 4-hr cytotoxicity assays. IFN-γ was superior to IFN-α in that respect. Among MLTC responder lymphocytes, tumor-reactive CTL proliferated more strongly in response to MZ-PC-2 cells treated with IFN-γ than to untreated tumor cells. Three CTL clones derived from MLTC were chosen for further analysis. They were CD3+, CD8+, TCR-α/β⁺ and behaved identically in all functional aspects tested. They all expressed the same TCR-β chain, indicating that they descended from a common precursor lymphocyte and were directed against the same antigen. According to antibody-inhibition experiments, BE-CTL recognized their targets via an HLA-B molecule carrying the Bw6 supertypic determinant. Irrespective of pre-incubation with IFN-γ, low levels of tumor-cell lysis, or none, were seen when MZ-PC-2 cells were kept in medium supplemented with autologous serum or serum pooled from healthy volunteers instead of FCS. Lysability was restored when TNF-α was added to human serum. Serum-free medium was found to enhance the susceptibility of MZ-PC-2 cells to lysis by autologous CTL.     

Induction of tolerance to a murine fibrosarcoma in two zones of dosage--the involvement of suppressor cells

Small size inocula (10(1)-10(3) cells) of cells from a syngeneic methylcholanthrene-induced fibrosarcoma (FSA) induced tolerance when injected s.c. into C3Hf mice. Mice were unable to respond to subsequent challenge with moderate, immunogenic doses of FSA. Tolerance was demonstrated in an in vivo transfer (Winn) assay and an in vitro tumour-specific TH cell assay. Low zone tolerance was associated with the presence of tumour-specific TS cells in the spleen. Moderate size inocula (10(4)-10(6) FSA cells) were immunogenic but larger cell doses (greater than 10(6)) were again tolerogenic. In the high zone, tolerance was associated with both tumour-specific TS cells and non T suppressor cells that were not tumour-specific. These results support the view that immunogenic tumours, as they grow from small cell numbers, might be able to escape host surveillance by specifically tolerizing the immune system. They also suggest that large tumour burdens can interfere with the host's immune response by inducing suppressor cells.     

Enhancement of anti-tumour immunity in syngeneic mice after MHC class II gene transfection.

The relationship between tumorigenicity and expression of MHC class II molecules in a class II-negative murine leukaemia cell line (LBC) was studied. Analysis of structural DNA sequences encoding MHC class II proteins was performed by Southern blot with DNA isolated from both the original LB tumour and LBC cell line, digested with EcoRI, BamHI and HindIII and hybridised with specific probes for I-A alpha d and I-A beta d chains. Similar patterns were obtained for LB, LBC and normal BALB/c lymphocytes. In vitro treatment with IFN-gamma (20 - 1000 IU ml-1) failed to induce the expression of MHC class II antigens in LBC cell line. LBC cells were tri-transfected by a liposome-mediated protocol with I-A alpha d, I-A beta d genes and pSV2neo. Cells were selected for growth in medium containing Geneticin (G418). Surviving transfectants were cloned and three I-A+ clones were obtained after 20 days (LBCT cells). Syngeneic mice inoculated with 1.0 x 10(3) LBCT (I-A+) cells failed to develop a tumour, whereas the DT50 of mice injected with 1.0 x 10(6) LBCT cells was three times the value for mice injected with LBC cells (I-A-). Furthermore, specific CTL response against tumour cells was significantly enhanced upon priming with irradiated LBC-transfected cells (27 +/- 2%) compared with irradiated LBC cells (15 +/- 1.5%) in a 4 h 51Cr-release assay. It is suggested that neoexpression of MHC class II molecules enhances anti-tumour response by transforming tumour cells into professional antigen-presenting cells (APCs), which may be used to improve tumour-specific immunity in the autologous host.     

A combination of interleukin-2 and 60 nm cationic supramolecular biovectors for the treatment of established tumours by subcutaneous or intranasal administration

The Supramolecular Biovector (SMBV) KY is a drug delivery nanocarrier which consists of a discretely sized, ionically charged, cross-linked polysaccharide core surrounded by a lipid membrane. We used the non-immunogenic spontaneous mammary adenocarcinoma TS/A tumour to test the efficacy on tumour growth of low (10(4) IU) or ultra-low (10(3) IU) doses of interleukin-2 (IL-2) adsorbed to these 60 nm cationic synthetic particles. In comparison with the progressive growth of TS/A cells in syngeneic mice, KY/IL-2 particles coinjected with TS/A cells or administered at a distance from the tumour, inhibited tumour growth while free IL-2, even at 10-100 times the dose used in the KY/IL-2 formulations, had no effect. Studies performed on implanted tumours (treatment at day 6 (D6)) showed that KY/IL-2 administered subcutaneously (s.c.) at five sites distant from the tumour (10(3) IL-2 IU per site) induced rejection of the implanted tumours. Six out of 10 mice were cured while the other four had residual tumours only. In the same experiment, free IL-2 induced only tumoral growth reduction. Protection induced by KY/IL-2 administered s.c. at five sites involved recruitment of a CD8(+) T cell response since nu/nu mice and CD8-depleted mice did not reject the tumours. Mice cured were protected significantly to completely against a rechallenge with TS/A tumour cells, and a systemic tumour-specific CTL activity was induced. Finally, we showed that repeated intranasal (i.n.) administration of KY/IL-2 (low-dose) also led to complete regression of pre-established tumours and partial protection from tumour rechallenge. We therefore suggest that, in contrast to free IL-2, a KY/IL-2 formulation could be used as a systemic immunostimulant leading to the eradication of non-immunogenic, established tumours.     

Antitumor activity of intraperitoneal immunotoxins in a nude mouse model of human malignant mesothelioma

Immunotoxins directed against human transferrin receptor have been evaluated in a nude mouse model of human malignant mesothelioma. Immunotoxins were constructed by linking ricin A chain to murine monoclonal antibodies reactive with the human transferrin receptor. A chain was obtained either by isolation from the parent toxin or by recombinant DNA techniques. These immunotoxins acted as potent in vitro cytotoxins against human malignant mesothelioma cells (H-MESO-1) (ID50, 2 X 10(-9) M). Cytotoxic potency and kinetics of cell kill were potentiated in vitro by the carboxylic ionophore monensin. For in vivo trials, nude mice were injected i.p. with 6-9 X 10(6) human malignant mesothelioma cells 24 h prior to the start of i.p. immunotoxin treatments. The survival of tumor-bearing mice was extended by 149-404%, representing a probable cell kill of 2-4 logs. Specificity of this antitransferrin receptor immunotoxin response was confirmed by the ineffectiveness of irrelevant control immunotoxins and blockade of specific immunotoxin action by excess free antibody. Monensin showed limited in vivo potentiation of immunotoxin effect, but a derivative formed by esterification of monensin with linoleic acid gave improved survival times over treatment with immunotoxin alone. Immunotoxins constructed with ricin A chain have significant tumoricidal activity in this model of regional antitumor therapy. These results may have direct relevance for treatment of i.p. malignancy in clinical settings.     

Effect of palytoxin and serotonin on murine tumor cells.

In vitro and in vivo effects of two fractions of a crude extract from the marine invertebrate Palythoa were investigated in Novikoff hepatoma and Ehrlich ascites tumor cells. These fractions, previously identified as serotonin and a partially purified toxin, inhibited thymidine incorporation into acid-precipitable material by tumor cells. Results from respiration studies indicated that the inhibitory effect was not toxic. The data suggested that: a) The partially purified toxin exerted its inhibitory effect at the membrane level; b) this inhibitory effect might have been accomplished through the Na+K+ transport mechanism, this conclusion being supported by data on the uptake of labeled amino acids by Ehrlich ascites tumor cells; and c) in an unconfirmed experiment, the partially purified toxin increased the survival time of 50% of 6 palytoxin-treated mice at least three times as long as 6 untreated control mice. All 12 mice had received 1 X 10(6) viable Ehrlich ascites tumor cells.     

An autologous T cell clone overcomes intra-melanoma heterogeneity for susceptibility to cell-mediated lysis by using multiple lytic mechanisms: in vitro and in vivo analysis.

An HLA-A2-specific cytotoxic T lymphocyte (CTL) clone (CTL49), capable of killing the HLA-A2-negative autologous melanoma (Me665/2) in a T cell receptor and MHC-independent fashion, lysed six of 16 Me665/2 tumour clones in short-term (4 and 18 hour) 51Cr-release assays. In long-term (96 hour) lytic assays, CTL49 could lyse all the 16 tumour clones. The lysis observed in the 96 hour assay could be enhanced by stimulating CTL49 with anti-CD3 monoclonal antibodies (MAb) and interleukin-2 (IL-2). Supernatants of anti-CD3- or antigen-stimulated CTL49, known to contain tumour necrosis factor (TNF) alpha and interferon (IFN)gamma, were also able to lyse all but one (665/2/51) of the tumour clones in 96 hour assays. Absence of lysis of tumour clone 2/51 by supernatants correlated with resistance of the same tumour clone to lysis by recombinant IFN-gamma plus TNF-alpha. Antibodies to TNF-alpha and, to a lesser extent, to IFN-gamma, reduced significantly the 96 hour lysis of Me2/9 and Me2/10, two of the tumour clones killed in long term but not in short term assays. Winn assays in nude mice revealed that CTL49, stimulated with anti-CD3-MAb plus IL-2, could abolish tumour cell growth when injected together with clones 2/9 or 2/10. These results indicate that intra-tumour heterogeneity for susceptibility to lysis can be overcome even by a single CTL clone providing that appropriate signals (i.e. anti-CD3-MAb and IL-2) are supplied to an effector able to mediate tumour cell lysis by multiple mechanisms.     

Anti-L-selectin monoclonal antibody treatment in mice enhances tumor growth by preventing CTL sensitization in peripheral lymph nodes draining the tumor area

To examine the in vivo contribution of L-selectin in the sensitization of tumor-specific CTL, we investigated the effects of treatment with the anti-L-selectin monoclonal antibody (MAb) MEL-14 on the immune response to Moloney-murine sarcoma virus (M-MSV)-induced tumors, which exhibit spontaneous regression following generation of a strong virus-specific CTL response. Daily systemic administration of MEL-14 for 10 days to M-MSV-injected mice gave rise to larger sarcomas that persisted for a longer time, compared with those arising in control mice injected with virus only. The enhanced tumor growth could not be attributed to cytotoxic activity on leukocytes by MEL-14 since no reduction in the total cell number was detected in peripheral blood and spleen of MAb-treated mice. Evaluation of the immunological response in MAb-treated animals revealed a strong reduction in the generation of virus-specific CTL precursors (CTLp) in tumor-draining peripheral lymph nodes (PLN) 10 and 15 days after M-MSV injection, while in spleen, where lymphocyte localization is independent of L-selectin expression, CTLp generation was only delayed. By day 20, when tumors had begun to regress, the CTLp number showed a marked increase in both spleen and local PLN, where naive recirculating CTL could now enter because L-selectin was no longer down-regulated or blocked by the injected MAb. Our findings indicate that functional inactivation of L-selectin by MEL-14 treatment prevented migration of naive L-selectin ⁺ CTL through high endothelial venules (HEV) and their accumulation in PLN draining the tumor area, thereby precluding the initiation of a tumor-specific CTL response that takes place primarily at this site. © 1996 Wiley-Liss, Inc.     

Release of soluble "blocking" and "suppressor" factors from normal lymphocytes treated with RNA from spleens of tumour-bearing mice.

RNA extracted from the spleens of tumour-bearing (TLRNA) and tumour-immune (ILRNA) mice was shown to transfer to normal lymphocytes (NL) the ability to produce factors that blocked specific tumour-cell cytotoxicity and mediated specific antibody-dependent cell cytotoxicity (ADCC). Aliquots of normal C3H mouse lymphocytes were treated with TLRNA or ILRNA and cultured in vitro in the absence of tumour antigen. Supernatants were collected at 24h intervals and tested in a microcytotoxicity assay for blocking and ADCC activities. Factors that inhibited tumour destruction by specifically sensitized lymphocytes at the level of both the tumour cells and effector cells were demonstrable in culture supernatants of NL pretreated with TLRNA (50 or 100 microgram/4 X 10(6) cells) but not ILRNA. However, treatment of NL with either RNA resulted in the production factors that mediated tumour-specific ADCC. Cytotoxicity testing and absorption studies of the tumour cell and a control cell (LM) indicated that factors mediating ADCC and blocking at the target-cell level were specific for the tumour. Suppressor activity at the effector-cell level was not absorbed by tumour cells and represents a separate and distinct mechanism of immunosuppression. These data indicate that RNA faithfully transfers "suppressive" as well as "positive" types of immune responses that have been reported previously for lymphocytes obtained directly from tumour-bearing and tumour-immune animals.     

Lysis of fresh human tumor cells by autologous large granular lymphocytes and T-lymphocytes: two distinct killing activities induced by coculture with autologous tumor

The specific and natural killer (NK)-restricted nature of autologous tumor killing by blood lymphocytes was studied in patients with carcinomatous pleural effusions. Large granular lymphocytes (LGL) and small T-lymphocytes were isolated by centrifugation on discontinuous Percoll density gradients. Tumor cells freshly isolated from pleural effusions of cancer patients were classified according to their susceptibility to purified LGL from normal donors in a 4-hour 51Cr release assay. Of 15 NK-sensitive tumors, 14 were lysed by fresh autologous LGL, whereas only 2 were killed by T-cells. Neither LGL nor T-cells were cytotoxic to NK-resistant autologous tumor. LGL and T-cells were then cultured in vitro with autologous tumor cells for 6 days. In 13 of 15 autologous mixed lymphocyte-tumor cultures (MLTC) NK-sensitive tumor-cultured LGL maintained their autotumor killing activity, whereas LGL cultured alone lost the activity. Depletion of high-affinity sheep erythrocyte-rosetting cells from Percoll-purified LGL resulted in an enrichment of effector cells. LGL from autologous MLTC were able to kill NK-susceptible allogeneic effusion tumor and K562 as were fresh LGL. No lysis of NK-resistant autologous tumor was observed with cultured LGL. In contrast, activation of T-cells in autologous MLTC resulted in the generation of autotumor killer cells in 10 of 15 NK-sensitive and 3 of 6 NK-resistant tumor samples. However, cultured T-cells were incapable of killing allogeneic tumor and K562. In autologous MLTC T-cells proliferated in response to autologous tumor, whereas no proliferation was observed in the culture of LGL. The enrichment of blasts from cultured T-cells on discontinuous Percoll gradients induced an augmentation of autotumor cytotoxicity, with no reactivity in blast-depleted, small, resting T-lymphocytes. These results indicated that 2 distinct types of autotumor-recognizing lymphocytes, LGL and T-cells, are present in the peripheral blood of cancer patients.     

Inhibition of angiogenesis and tumour growth by VEGF121-toxin conjugate: differential effect on proliferating endothelial cells

Vascular endothelial growth factor (VEGF) plays an important role in tumour angiogenesis. VEGF binds to tyrosine kinase receptors, which are expressed almost exclusively on tumour endothelium. Therefore, VEGF can be used to target toxin molecules to tumour vessels for anti-angiogenic therapy. However, recent evidence suggests that VEGF can also bind in an isoform-specific fashion to a newly identified neuropilin-1 (NP-1) receptor. NP-1 is widely expressed in normal tissue and presents a potential target for unwanted toxicity. As a consequence, we investigated whether the VEGF121 isoform, which lacks the NP-1 binding domain, could be used to target toxin polypeptides to tumour vasculature. Treatment of endothelial cells with a VEGF121-diphtheria toxin (DT385) conjugate selectively inhibited proliferating endothelial cells, whereas confluent cultures were completely resistant to the construct. In addition, VEGF121-DT385 conjugate treatment completely prevented tumour cell induced angiogenesis in vivo. Most importantly, the conjugate inhibited tumour growth in athymic mice and induced tumour-specific vascular damage. There was also no apparent toxicity associated with the treatment. Our results suggest that proliferating endothelial cells are highly sensitive to VEGF121-toxin conjugates and that the binding to NP-1 receptors is not necessary for efficient inhibition of tumour growth.     

Therapeutic effect of interleukin 12 on mouse haemangiosarcomas is not associated with an increased anti-tumour cytotoxic T-lymphocyte activity.

In syngeneic mice, the H5V polyoma middle-T oncogene-transformed endothelioma cell line induces Kaposi''s sarcoma-like cavernous haemangiomas that regress transiently, probably because of an anti-tumour immune response, but eventually grow progressively and kill the host. To evaluate the generation of tumour-specific cytotoxic T lymphocytes (CTLs), spleen cells of tumour-bearing mice were restimulated with irradiated H5V cells in mixed leucocyte-tumour cell cultures. Tumour-specific CTLs were demonstrable only when low numbers of H5V stimulator cells were used (<1 H5V cell per 50 splenocytes). We found that H5V cells secrete immunosuppressive mediators because CTL generation was blocked when H5V cells culture supernatants were added to allogeneic mixed leucocyte cultures. As numerous tumour-derived immunosuppressive mediators may interfere with interleukin 12 (IL-12) production, we tested whether IL-12 treatment of the tumour-bearing mice would augment their immune response and thus suppress tumour growth. Indeed, IL-12 inhibited tumour growth and prevented mortality, but did not increase anti-H5V CTL generation either in vitro or in vivo. Moreover, the anti-tumour activity in IL-12-treated mice was abrogated by anti-interferon (IFN)-gamma monoclonal antibody (MAb) co-administration. These results strongly suggest that the anti-tumour effect of IL-12 is principally mediated by IFN-gamma release that in turn blocks H5V cell proliferation and induces the release of factors that suppress angiogenesis.     

Antitumour responses induced by short-term pretreatment with tumour cells.

The injection (s.c. or i.p.) of 10(6) live or lethally irradiated methylcholanthrene-induced fibrosarcoma cells into CBA/Ca mice one or 2 days before i.v. challenge with the same tumour inhibited the formation of artificial lung tumour metastases. In addition, it also frequently enhanced the cytostatic effect of peritoneal exudate cells on monolayers of the same tumour. The effects on lung tumour metastasis were not noted if X-irradiated tumour was injected i.v., or if s.c. administration was delayed until one day after i.v. challenge. Similar effects on tumour growth were also observed in C3Hf/Bu mice and (CBA/Ca x A/HeJ) F1 hybrids which were pretreated (s.c.) with tumour shortly before i.v. challenge with the same tumour. Further studies in CBA/Ca mice suggested that the protective effect was tumour-specific, for the growth of i.v. injected tumour was not significantly inhibited by pretreatement with a number of other MC-induced or spontaneous tumours from the same and different strains.     

Studies on the antigenicity of radiation-induced murine osteosarcomata

The immunogenicities of 15 murine osteosarcomata induced with a bone seeking radioisotope (⁹⁰Sr) in normal and chimaeric CBA mice were studied. Attempts were made to induce tumour-specific immunity in syngeneic mice by treatment with x-irradiated (15,000 rad) tumour or surgical excision of developing subcutaneous tumour grafts. Resistance was evoked against 6 tumours and this was relatively weak. With the remaining tumours, no resistance against the immunizing tumour could be demonstrated, even though the transplantation tests were made highly sensitive by the use of inocula of as few as 2 × 10³ cells in pre-irradiated (400 rad) hosts. Sera from mice immunized against each of the tumours were tested against viable cells of the immunizing tumour by indirect immunofluorescence. In no instance did tumour antisera give a convincing reaction with tumour cells although alloantisera raised by hyperimmunization of H-2 identical and H-2 different donors with osteosarcomata consistently gave strongly positive reactions. The results are interpreted as illustrating the weak tumour specific antigenicity of radiation-induced murine osteosarcomata. The possibility that antigenic deficiency is a consequence of immunosurveillance in this tumour system is discussed.     

Serological aspects of rat tumour xenograft growth in athymic nude mice.

The serum of athymic nude mice bearing rat tumour xenografts has been examined for tumour-specific antigen. With a sarcoma and a hepatoma, tumour-specific antigen expression continued in xenograft growths, and sera of tumour-bearing mice contained free antigen, assayed by its ability to neutralise reactivity of tumour-immune rat sera against tumour target cells in an indirect membrane-immunofluorescence test. In contrast, no anti-rat antibody was detectable in sera of mice bearing the xenografts, or rejecting cells injected in admixture with BCG.