Specific eradication of micrometastases by transfer of tumour-immune T cells from major-histocompatibility-complex congenic mice

Summary DBA/2 (H-2^d) mice bearing a transplanted highly metastatic lymphoma (ESb) in a state of widely disseminated disease could be successfully treated by a combination of surgery (removal of the local tumour), irradiation (5 Gy) and adoptive immunotherapy. The immunotherapy was achieved by transfer of anti-ESb-immune spleen cells from B10.D2 mice, which express the same major histocompatibility complex (MHC) molecules as DBA/2. In contrast, anti-ESb-immune cells from MHC-disparate C57BL/6 mice did not confer protective immunity. The B10.D2 anti-ESb-immune T cells contain two types of cytolytic specificity as detected by limiting-dilution analysis: (1) clones with specificity for the ESb-tumour-associated transplantation antigen (TATA) (at low frequency), and (b) clones with specificity for minor DBA/2 histocompatibility (H) antigens (at high frequency). Immune B10.D2 cells raised against different tumour lines or against TATA^– ESb tumour variants did not confer the 100% protection seen with immune cells against ESb TATA⁺ cells. Finally we demonstrate that the allogeneic immune cells are more potent in terms of protective immunity than corresponding syngeneic immune cells. The data suggest that the strong graft-versus-leukemia effect with immune T cells from allogeneic MHC-identical but not from MHC-disparate mice was due to T cells with MHC-restricted specificity for an ESb-associated TATA. A graft-versus-host reactivity that developed much later and could not be prevented was most likely due to T cells sensitized against normal minor H antigens of the host. Our results are of potential relevance for allogeneic bone marrow transplantation and adoptive immunotherapy protocols.     

Fate of H2-activated T lymphocytes in syngeneic hosts. III. Differentiation into long-lived recirculating memory cells.

Memory to H2 determinants was studied with an adoptive transfer system using a population of H2-activated blast T cells (T.TDL) obtained from thoracic duct lymph of irradiated F₁ hybrid mice injected with parental strain T cells. CBA T.TDL activated either to DBA/2 or C57BL determinants were transferred to syngeneic “B” mice. Thoracic duct lymphocytes (TDL) were obtained from the recipients 4–6 weeks later and tested for their capacity to produce (a) a graft-versus-host (GVH) reaction, (b) a mixed lymphocyte reaction (MLR) (measured by an in vivo technique) and (c) allograft rejection (suppression of the growth of allogeneic tumour cells in vivo). Control experiments involved testing the function of TDL obtained from “B” mice preinjected with TDL or no cells.TDL from “B” mice injected with TDL (passaged TDL) gave strong MLR and GVH reactions to both DBA/2 and C57BL determinants. Passaged T.TDL activated to C57BL antigens gave intermediate MLR and GVH reactions to the specific (C57BL) determinants but only very low responses to third-party (DBA/2) determinants; reciprocal results were obtained with passaged T.TDL activated to DBA/2 determinants. TDL from “B” mice given no cells failed to respond to either set of determinants.Since the responses by the passaged T.TDL did not exceed those by passaged TDL there was no evidence that adoptive transfer of T.TDL had conferred to the recipients a state of memory to either MLR or GVH determinants. Adoptive transfer did, however, lead to qualitative changes in the properties of T.TDL since, before transfer, they were unable to evoke GVH reactions or produce an MLR of normal kinetics.Passaged T.TDL were far superior to passaged TDL at suppressing the growth of allogeneic tumour cells. The protection was specific since protection against DBA/2 tumour cells was, cell for cell, 5–10 fold more effective with passaged T.TDL activated to DBA/2 determinants than with cells activated to C57BL determinants. No protection was observed with cells treated with anti-θ serum. The protective cells appeared to be precursors of effector cells rather than effector cells per se since they failed to lyse the tumour cells in vitro. These data suggest therefore that the descendants of T.TDL which survived after transfer to “B” mice were highly enriched in long-lived recirculating T lymphocytes reactive to determinants expressed by specific tumour allografts.     

Immunotherapy success in prophylaxis cannot predict therapy: prime-boost vaccination against the 5T4 oncofoetal antigen

We have investigated the tumour therapeutic efficacy of homologous and heterologous prime-boost vaccine strategies against the 5T4 oncofoetal antigen, using both replication defective adenovirus expressing human 5T4 (Ad5T4), and retrovirally transduced DC lines (DCh5T4) in a subcutaneous B16 melanoma model (B16h5T4). In naïve mice we show that all vaccine combinations tested can provide significant tumour growth delay. While DCh5T4/Adh5T4 sequence is the best prophylactic regimen (P > 0.0001), it does not demonstrate any therapeutic efficacy in mice with established tumours. In active therapy the Adh5T4/DCh5T4 vaccination sequence is the best treatment regimen (P = 0.0045). In active therapy, we demonstrate that B16h5T4 tumour growth per se induces Th2 polarising immune responses against 5T4, and the success of subsequent vaccination is dependant on altering the polarizing immune responses from Th2 to Th1. We show that the first immunization with Adh5T4 can condition the mice to induce 5T4 specific Th1 immune responses, which can be sustained and subsequently boosted with DCh5T4. In contrast immunisation with DCh5T4 augments Th2 immune responses, such that a subsequent vaccination with Adh5T4 cannot rescue tumour growth. In this case the depletion of CD25⁺ regulatory cells after tumour challenge but before immunization can restore therapeutic efficacy. This study highlights that all vaccine vectors are not equal at generating TAA immune responses; in tumour bearing mice the capability of different vaccines to activate the most appropriate anti-tumour immune responses is greatly altered compared to what is found in naïve mice.     

Superiority of the ear pinna over a subcutaneous tumour inoculation site for induction of a Th1-type cytokine response

 This study examines whether a correlation may be found between Th1- or Th2-type cytokine responses and resistance or susceptibility to tumour growth. Cytokine profiles were investigated in a well-defined mouse tumour model in which the injection site and the genetic background determine the phenotype of either tumour resistance or tumour susceptibility. DBA/2-derived ESb lymphoma variant cells with high metastatic capacity were inoculated into syngeneic mice either s.c., where they grow and metastasize, or into the ear pinna (i.e.), where they do not grow because of induction of protective immunity. Alternatively, the tumour cells were injected s.c. or i.e. into allogeneic B10.D2 mice, which are resistant to the tumour although they are identical at the MHC locus. Between 1 and 10 days after tumour cell injection the spleen-derived mRNA was tested for cytokine gene expression or the spleen cells were analysed by FACScan for T cell activation. The strongest cytokine response was observed in i.e. inoculated B10.D2 mice. This was characterized by an early (days 2–3) peak of interferon γ (INF-γ), interleukin-2 (IL-2), IL-2 receptor α (IL-2Rα) and IL-4. The cytokine mRNA response of i.e. inoculated DBA/2 mice was quite similar except that no IFN-γ could be detected. In s.c. inoculated B10.D2 mice, the IL-2, IL-2Rα and IFN-γ responses were weaker than after i.e. injection while the IL-4 response was comparable. The most striking difference between these cytokine profiles from tumour-resistant mice and those of s.c. inoculated tumour-susceptible DBA/2 mice was a delay in the latter in the IL-2, IL-2Rα and IFN-γ responses and the observation that the IL-4 response was not down-regulated. The persisting IL-4 response could down-regulate a Th1-type response and thereby explain tumour susceptibility as a consequence of host conditioning. Received: 4 September 1997 / Accepted: 2 October 1997     

5,6-Dimethylxanthenone-4-acetic acid treatment of a non-immunogenic tumour does not synergize with active or passive CD8+ T-cell immunotherapy

The chemotherapeutic drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) inhibits intratumoural blood flow, causing hypoxia, haemorrhagic necrosis, vascular collapse and tumour cell death. Production of TNF-alpha and IFN is also induced, causing local inflammation and activation of immune cells including CD8+ T cells. We used the tumour cell line LL-LCMV, which expresses the gp33 epitope of lymphocytic choriomeningitis virus in a non-immunogenic form, to investigate whether tumour cell death caused by treatment with DMXAA may improve the success of tumour immunotherapy mediated by CD8+ T cells. Treatment with DMXAA was effective at reducing the size of LL-LCMV tumours. However, compared to normal mice, tumour reduction was no more marked or sustained in mice carrying high numbers of naive, tumour-specific CD8+ T cells. The antitumour effect of activated CD8+ T cells was also not affected by DMXAA treatment. Tumour-specific CD8+ T cells activated in vivo by immunization with dendritic cells and specific tumour peptide antigen, or generated in vitro and adoptively transferred into tumour-bearing mice by i.v. injection, did not improve or sustain the reduction in tumour size induced by DMXAA treatment. We conclude that the presence of high numbers of naive CD8+ T cells, or immunotherapies leading to CD8+ T-cell activation, do not synergize with the tumour cell death and local inflammation induced by DMXAA treatment. It is possible that this lack of synergism may result from both treatments inducing activation of CD8+ T cells and that treatments that activate different populations of immune cells may achieve better success.     

Protein dimerization between Lmo2 (Rbtn2) and Tal1 alters thymocyte development and potentiates T cell tumorigenesis in transgenic mice.

The LMO2 and TAL1 genes were first identified via chromosomal translocations and later found to encode proteins that interact during normal erythroid development. Some T cell leukaemia patients have chromosomal abnormalities involving both genes, implying that LMO2 and TAL1 act synergistically to promote tumorigenesis after their inappropriate co-expression. To test this hypothesis, transgenic mice were made which co-express Lmo2 and Tal1 genes in T cells. Dimers of Lmo2 and Tal1 proteins were formed in thymocytes of double but not single transgenic mice. Furthermore, thymuses of double transgenic mice were almost completely populated by immature T cells from birth, and these mice develop T cell tumours approximately 3 months earlier than those with only the Lmo2 transgene. Thus interaction between these two proteins can alter T cell development and potentiate tumorigenesis. The data also provide formal proof that TAL1 is an oncogene, apparently acting as a tumour promoter in this system.     

Growth of Theileria annulata and Theileria parva macroschizont-infected bovine cells in immunodeficient mice: effect of irradiation and tumour load on lymphocyte subsets.

Bovine cells infected with macroschizonts of the protozoan parasites Theileria annulata and Theileria parva formed solid tumours when injected into irradiated Balb/c and irradiated Balb/c nude mice. T. annulata tumours grew more vigorously than T. parva tumours, when initiated with similar doses of infected cells in mice exposed to the same doses of gamma-irradiation. In irradiated Balb/c mice, tumours of both species of parasites began to regress 2-3 weeks after injection of cells but grew without regression in irradiated Balb/c nude mice. Haemorrhage and necrosis of tumours, induced by macrophages and neutrophils, were seen in both mouse strains but were insufficient to cause regression in Balb/c nude mice. Theileria-infected bovine cells failed to establish in C57 beige mice, which lack functional natural killer (NK) cells. Flow cytometry, using monoclonal antibodies to murine leukocyte/lymphocyte antigens, showed that the radiation dose required to allow establishment of T. annulata tumours in Balb/c mice caused a severe depletion of splenic lymphocytes. B cells, helper T and cytotoxic T cells showed differing levels of susceptibility to irradiation. The presence of a tumour promoted the recovery of lymphocyte populations: this recovery was accompanied by destruction of the tumour.     

Enhanced immune response induced by P5 HER2/neu‐derived peptide‐pulsed dendritic cells as a preventive cancer vaccine

Dendritic cells are special and powerful antigen‐presenting cells that can induce primary immune responses against tumour‐associated antigens. They can present antigens via both MHC‐I and MHC‐II, so they have the ability to stimulate both cytotoxic T lymphocytes and T helper cells. Furthermore, CD8⁺ cytotoxic T lymphocytes require activation by CD4⁺ T cells. This requires a CD4⁺T cell activator molecule, of which PADRE is one of the best. We chose an approach to use both of these important arms of the immune system. We prepared dendritic cells from mouse bone marrow, loaded them with our target peptides (P5 peptide alone or P5 + PADRE), and then injected these pulsed dendritic cells alone or in combination with CpG‐ODN (as adjuvant) into BALB/C mice. After the last boosting dose, mice were inoculated with TUBO cells, which overexpress HER2/neu. Two weeks after the tumour cell injection, immunological tests were performed on splenocyte suspensions, and the remaining mice were evaluated for tumour growth and survival. Our data indicate the formulation that contains PADRE plus P5 loaded onto DC in combination with CpG‐ODN was the most effective formulation at inducing immune responses. Interferon production in CD4⁺ and CD8⁺ gated cells, cytotoxicity rates of target cells and mice survival were all significantly greater in this group than in controls, and all the mice in this group were tumour‐free throughout the experiment. Based on our results and the role of HER2/neu as a candidate in human immunotherapy, this approach may be an effective cancer treatment.     

Suppressive effect of adrenalectomy on growth of L1210 leukemic cells in ascites

This study was designed to evaluate the effect of adrenalectomy on growth of L1210 leukemic cells in ascites of BDF1 mice. Varying doses of 1.5 x 10(4), 5.0 x 10(5), and 1.5 x 10(6) viable tumour cells were inoculated intraperitoneally into groups of either adrenalectomized or sham-operated mice. At days 4 to 7 after the inoculation, adrenalectomized mice inoculated with 1.5 x 10(4) or 5.0 x 10(5) tumour cells had a smaller number of tumour cells in ascites than sham-operated controls. However, after inoculation of 1.5 x 10(6) cells, no significant differences were found at days 2 to 4 between adrenalectomized and sham-operated mice. The growth retardation by adrenalectomy was not observed in adrenalectomized mice supplemented with 4 or 6 micrograms dexamethasone per day per mouse. It suggested that the ablation of glucocorticoids was at least partially responsible for the growth retardation observed in adrenalectomized mice. Cell kinetic analysis revealed that the difference in a potential doubling time could not explain these results. Tumour retention in the peritoneal cavity was measured using [125I]-iododeoxyuridine-labelled tumour cells as a tracer. At days 4 to 6 after inoculation of 5.0 x 10(5) labelled cells, radioactivity in the peritoneal cavity in adrenalectomized mice was about 70 per cent of that in sham-operated mice. This ratio was almost equivalent to the ratio of the number of cells in ascites of adrenalectomized mice to that of sham-operated ones. Consequently, growth retardation observed in adrenalectomized mice resulted from an increase in tumour cell migration and/or in tumour cell death, but not from an increase in doubling time.     

Histological analysis of IL-2 induced regression of murine solid SL2-tumors

When DBA/2 mice are inoculated both intraperitoneally (i.p.) and subcutaneously (s.c.) with syngeneic SL2 lymphoma cells and treated i.p. on day 10–14 with 20,000 units IL-2/day, about 50% of the mice reject both the ascitic tumour and the s.c. tumour. During IL-2 therapy large areas of necrosis appear in the solid SL2 tumours between day 12 and 15. Immunohistochemical studies show that only a small number of infiltrating cells is present in the tumours. The percentage of macrophages (MHC-II⁺)in the tumours is about 1 and the percentage of T-lymphocytes (-TCR⁺) about 0.5. No differences in the numbers of infiltrating cells are seen in untreated and IL-2 treated tumour bearing mice. The tumoursurrounding infiltrate consists mainly of mononuclear cells: about 50% macrophages, 20% CD8⁺ cells, and 15% CD4⁺ cells. No tumour-infiltrating cells were found that express the IL-2 receptor.We conclude that direct cytotoxic activity of tumour infiltrating cells cannot account for the rapid occurrence of necrosis.When L3T4⁺ cells were eliminated by treating the mice with-L3T4 monoclonal antibodies before tumor inoculation and treatment with rIL-2, tumor eradication did not occur. So, L3T4⁺ helper T-cells are essential for IL-2-mediated tumour regression. Exogenous rIL-2 is not directly responsible for the induced tumour regression. A significant stagnation of intratumoural bloodflow is observed after histological analysis; yet it still needs to be determined whether this is the primary cause or consequence of the observed necrosis.Abbreviations BSA bovine serum albumin - CTL cytotoxic T-lymphocyte - FACS fluorescence activated cell sorter - HE haematoxylin and eosin - IFN interferon - IL-2 interleukin-2 - IL-2R interleukin-2 receptor - i.p. intraperitoneal(ly) - i.V. intravenous(ly) - LAK lymphokine-activated killer - MHC major histocompatibility complex - PBS phosphate buffered saline - s.c. subcutaneous(ly) - TCR T-cell receptor - TNF tumour necrosis factor     

The production of a 70 kDa heat shock protein by Toxoplasma gondii RH strain in immunocompromised mice

A 70 kDa heat shock protein (HSP70) has been reported previously to be strongly expressed in virulent Toxoplasma gondii strains taken from immunocompetent mice but it is poorly expressed by virulent parasites in mice immunocompromised by treatment with cortisone acetate or by virulent parasites cultured in vitro. Immune factors such as interferon-γ, tumour necrosis factor and reactive nitrogen intermediates derived from nitric oxide are known to be important inducers of HSP70 production and are also known to be produced during the immune response to acute T. gondii infection. The ability of these immune factors to induce T. gondii HSP70 production was tested by analysing HSP70 production in tachyzoites of the virulent RH strain of T. gondii recovered from mice deficient in: (1) T cells (nude mice); (2) T and B cells (SCID mice); (3) interferon-γ receptors (interferon-γ receptor knockout mice); and (4) tumour necrosis factor receptors (tumour necrosis factor receptor knockout mice). Parasites from nude and SCID mice produced as much HSP70 as immunocompetent mice. Likewise, T. gondii tachyzoites from mice lacking receptors for interferon-γ or tumour necrosis factor produced HSP70 in quantities similar to wild-type mice. The ability to produce reactive nitrogen intermediates in response to T. gondii infection, as detected by elevated levels of nitrate and nitrite in sera, was normal in tumour necrosis factor receptor knockout mice but was completely lacking in interferon-γ receptor knockout mice, indicating that reactive nitrogen intermediates are also not responsible for induction of parasite HSP70. Thus, immune factors that induce HSP70 production in mammalian cells do not appear to play primary roles in inducing HSP70 production by T. gondii.     

Mechanisms of tumour rejection in the murine DBA/2-SL2 concomitant immunity system

Summary The mechanisms of rejection of secondary tumours were studied in concomitant immunity using transplants of standard-size solid SL2 tumours in syngeneic DBA/2 mice. In such a system primary tumour implants are not rejected, in contrast to secondary tumour implants. The second tumour was mainly rejected 2–4 days after implantation. Both primary tumour and secondary tumour implants (which are rejected) contained hardly any lymphocyte infiltrate, whereas, 2–4 days after implantation they contained 40%–50% macrophages, which were cytotoxic in vitro. Transfer (s.c.) of these tumours to naive mice showed that cellular infiltrates in the secondary implants did not always cause tumour rejection. Serum collected on day 4 after implantation of the secondary tumour was cytotoxic to SL2 tumour cells in vitro, whereas serum from mice with only primary implants was not cytotoxic on day 4 after implantation. Preliminary characterization of this cytotoxic factor showed that it was heat-labile, as cytotoxicity disappeared after 30 min at 56°C, the molecular mass of the factor was higher than 100 kDa, and it was not IgG. We hypothesize that secondary tumours in the DBA/2-SL2 concomitant immunity system are rejected mainly between 2 and 4 days after implantation as a result of the combined action of cytotoxic serum and the presence of 40%–50% cytotoxic macrophages. The primary tumour is not rejected at 2–4 days after implantation as there is no cytotoxic factor.     

T cell subsets in (responder x nonresponder)F1 mice regulating antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine (GAT)

Immune responses to GAT are controlled by H-2-linked Ir genes; soluble GAT stimulates antibody responses in responder mice (H-2b) but not in nonresponder mice (H-2q). In nonresponder mice, soluble GAT stimulates suppressor T cells that preempt function of helper T cells. After immunization with soluble GAT, spleen cells from (responder x nonresponder: H-2b X H-2q)F1 mice develop antibody responses to responder H-2b GAT-M phi but not to nonresponder H-2q GAT-M phi. This failure of immune F1 spleen cells to respond is due to an active suppressor T cell mechanism that is activated by H-2q, but not H-2b, GAT-M phi and involves two regulatory T cell subsets. Suppressor-inducer T cells are immune radiosensitive Lyt-1 +2-, I-A-, I-J+, Qa-1+ cells. Suppressor-effector T cells can be derived from virgin or immune spleens and are radiosensitive Lyt-1-2+, I-A-, I-J+, Qa-1+ cells. This suppressor mechanism can suppress responses of virgin or immune F1 helper T cells and B cells. Helper T cells specific for H-2b GAT-M phi are easily detected in F1 mice after immunization with soluble GAT; helper T cells specific for H-2q GAT-M phi are demonstrated after elimination of the suppressor-inducer and -effector cells. These helper T cells are radioresistant Lyt-1+2-, I-A+, I-J-, Qa-1- cells. These data indicate that the Ir gene defect in responses to GAT is not due to a failure of nonresponder M phi to present GAT and most likely is not due to a defective T cell repertoire, because the relevant helper T cells are primed in F1 mice by soluble GAT and can be demonstrated when suppressor cells are removed. These data are discussed in the context of mechanisms for expression of Ir gene function in responses to GAT, especially the balance between stimulation of helper vs suppressor T cells.     

A novel tumour model system for the study of long-term protective immunity and immune T cell memory

We present a novel non-transgenic system to be used for studies on anti-tumour adoptive immunotherapy (ADI) and long-term T cell memory. Tumour-reactive donor immune cells against lacZ-transfected syngeneic tumour cells (ESbL-Gal) were generated from a naíve T cell repertoire in DBA/2 mice by a well-established priming/restimulation protocol, and transferred to tumour-inoculated athymic nu/nu mice. The donor immune cells efficiently mediated protective anti-tumour immunity involving both CD4(+) and CD8(+) T cells, and anti-metastatic effects were stronger in 4.5 Gy pre-irradiated than in non-irradiated tumour-inoculated hosts. Long-term persistence of beta-galactosidase (Gal)-specific T cells was shown ex vivo by tetramer staining of CD8(+) T cells specific for an immunodominant Gal epitope. Resistance of treated nu/nu mice against tumour rechallenge revealed the existence of long-term protective immune memory.     

Non-cytotoxic activity of pyran copolymer-induced macrophages associated with potentiation of tumour vaccine in recipient mice

Summary Mice inoculated with both L1210 murine tumour vaccine and pyran copolymer were more resistant to L1210 than those inoculated with either of these agents alone. Rabbit anti-mouse thymocyte globulin and silica reduced the augmented resistance of these mice, suggesting the involvement of activated anti-tumour T cells and macrophages in the augmented resistance. We studied the activation of these two cells separately and examined the possible contribution of pyran copolymer-induced peritoneal cells to the augmented resistance to an inoculation of live tumour. Pyran copolymer-induced peritoneal cells endowed the tumour vaccine-primed mice, but not unprimed mice, with resistance to implanted L1210 and, among those peritoneal cell populations, macrophages but not T cells were responsible for this effect since the activity was associated with a cell population which was (1) adherent to nylon wool columns, (2) sensitive to silica and (3) insensitive to anti-Thy 1.2 antibody plus complement. The pyran copolymer-induced peritoneal cells had very little antiproliferative activity when tested against L1210 in vitro and mice inoculated with these peritoneal cells did not survive a challenge of live L1210 cells much longer (<1 day) than L1210 inoculated control mice. Furthermore, the survival of L1210 vaccine-primed mice inoculated with one-tenth the amount of live L1210 (10²) was still much shorter than that of mice primed with L1210 vaccine plus pyran copolymer and challenged with ten times as many (10³) live L1210 cells. Therefore, direct tumouricidal activity was probably not a major factor in the in vivo immunological augmenting activity of the pyran copolymer-induced macrophages.     

Mechanisms of BCG action

Summary Donor mice were treated IV with BCG and after various time intervals the spleens from these animals were injected into syngeneic recipients which were simultaneously challenged with an allogeneic tumour. The spleen cells from the BCG-treated donors, but not untreated donors, conferred on the recipients an ability to induce a potentiated CMC reaction against the tumour. The transference of BCG-induced potentiating activity could not be explained by the transference of viable BCG organisms, but was mediated by a cell that was anti-Thy.1-sensitive, silica-resistant, plastic-nonadherent, and nylon wool-adherent, and was sensitive in vivo to anti-thymocyte serum but resistant to hydrocortisone. By the use of congenic strains of mice that differed at the Thy.1 allele, it was shown that the cells responsible were not precursors of the cytotoxic lymphocytes but were cells that produced an amplification of the response of the recipient host's precursor cytotoxic T cells.     

The use of cytotoxic T cells in the regulation of tumour growth in syngeneic mice

Summary Normal C57BL/6 (B6) spleen cells were cultured with syngeneic EL4 tumour cells, expanded in IL2-containing medium, and tested for anti-tumour activity in vitro and in vivo. The activated cells were highly cytotoxic for EL4 and to a lesser degree killed syngeneic B6 blasts and allogeneic (D2) P815 tumour cells. B6 or BDF1 mice that received these cultured cells by IP injection cleared ¹²⁵IUdR-labelled EL4 cells faster than untreated mice. However, this enhanced clearance was evident only 7–12 days after injection. Since the injected cells had a short half-life (<10% remaining after 48 h) the effect of these cells in vivo was most probably due to the activation of the host's immune system. Mice that received cultured cells survived significantly longer than untreated mice following a lethal dose of EL4 cells. Cultured cells were much more effective in prolonging survival when used in conjunction with cyclophosphamide (CY). In animals receiving either cultured cells with or without CY or CY alone tumour clearance was markedly enhanced 7–12 days after injection.When challenged with a small dose of EL4 tumour cells (1×10⁴ SC per mouse) three of ten B6 mice treated with B6 anti-EL4 cultured cells were able to survive indefinitely. The frequency of CTL precursors to EL4 from the spleen cells of these surviving animals was about five-fold higher than that of normal spleen cells. Furthermore, CTL derived from primed spleen cells were more specific for EL4 than those derived from normal spleen cells.Abbreviations B6 C57BL/6J - BDF1 (C57BL/6J×DBA/2J) F1 - ConA SN concanavalin A supernatant - CTL cytotoxic T lymphocytes - CTL-P cytotoxic T-lymphocyte precursors - CY cyclophosphamide - E/T effector-to-target ratio - IL2 interleukin 2 - IP intraperitoneal - IUdR iododeoxyuridine - IV intravenous - LPS lipopolysaccharide - MST mean survival time     

Recruitment of helper T cells for induction of tumour rejection by cytolytic T lymphocytes

Immunotherapy of cancer could be possible in cases in which competent effector T cells can be induced. Such an approach depends on expression of tumour-specific antigens by the tumour cells and on the availability of sufficient costimulatory support for activation of cytotoxic T lymphocytes. Here, a strategy for helper T cell recruitment for induction of tumour-specific cytotoxic immune responses is presented. Allogenic MHC class II molecules were introduced into tumour cells by cell fusion. These hybrid cells, when injected into mice, induced rejection of an established tumour. The contribution of CD4-expressing helper T cells in the induction phase and of CD8-expressing T cells in the effector phase of the immune response was demonstrated. The approach described could be applicable to cases in which a suitable tumour antigen is present but not identified; it employs regulatory interactions that govern physiological immune responses and is designed to be minimally invasive.     

Cell kinetic studies of endothelial cells in the adenocarcinoma EO 771 and the effect of cyclophosphamide

Cell kinetic studies of endothelial cells in the adenocarcinoma EO 771 growing in C57bl/6j mice and after transplantation into Balb/c-nu/nu mice, as well as of the effect of cyclophosphamide treatment have been carried out. The³H-thymidine labelling index of endothelial cells decreases from about 8% 3–6 days after tumour inoculation to about 3% at 18 days. This decrease parallels that of the labelling index of tumour cells, i.e. there is a positive correlation between the labelling index of endothelial cells and that of tumour cells. The labelling index of endothelial cells in the tumour periphery is two to three times as high as that in the tumour centre reflecting corresponding differences in the rate of proliferation. There is no difference in the proliferation of endothelial cells whether the tumour grows in C57bl/6j or in Balb/c-nu/nu mice. After treatment with cyclophosphamide the labelling index of endothelial cells decreases within 2 days to 1–2% and remains that low despite regrowth of the tumour with increased tumour cell proliferation, indicating that tumour relapse does not depend on tumour angiogenesis.     

Two defects in old New Zealand Black mice are involved in the loss of low-dose paralysis to type III pneumococcal polysaccharide

Treatment of normal mice with a subimmunogenic dose of type III pneumococcal polysaccharide (SSS-III) results in the development of an antigen-specific state of unresponsiveness termed low-dose paralysis. This unresponsiveness is mediated by T suppressor cells and can be transferred by Lyt-2+ T cells, but not by L3T4+ T cells, obtained 18 hr after priming. As autoimmune New Zealand Black (NZB) mice age, there is a progressive decrease in low-dose paralysis to SSS-III. The defect in older NZB mice resulting in decreased suppressive activity was investigated by transferring primed Lyt-2+ T cells from young into old mice, and vice versa. Enlarged Lyt-2+ T cells from old NZB mice could not suppress the SSS-III response of young recipients. However, Lyt-2+ T cells of normal cell size were efficient in inhibiting the antibody response upon transfer. Primed Lyt-2+ T cells from young NZB mice did not affect the response of old recipients, but effectively suppressed the response of young mice. These results suggest that there are two defects involved in the decline of low-dose paralysis to SSS-III in aging NZB mice: Enlarged Lyt-2+ T cells may lose their ability to function as mediators of suppression; and B cells may become resistant to T cell-mediated suppression.