Darbie L. Maccubbin

Protective specific immunity induced by doxorubicin plus TNF-α combination treatment of EL4 lymphoma-bearing C57BL/6 mice

The therapeutic efficacy of a single (day 8), moderate dose (4 mg/kg, i.v.) of doxorubicin (DOX, Adriamycin) combined with recombinant human TNF‐α (3 different doses and 5 different schedules, i.v.) was evaluated in C57BL/6 mice bearing an implant (s.c.) of the DOX‐sensitive, TNF‐α‐resistant EL4 lymphoma. In parallel to monitoring survival, the levels of several host anti‐tumor cytolytic effector functions of splenocytes and thymocytes were evaluated throughout the treatment period and in long‐term survivors (LTS). DOX treatment alone resulted in a moderate (approx. 20%) increase in life span but no cures. TNF‐α alone, at any tested dose or schedule, had little or no positive effect on survival. The combinations of DOX and TNF‐α were only slightly better than DOX alone with respect to the time to death of mice that died (approx. 29% increase); however, each of the combinations involving 1,000 U TNF‐α/injection produced a fraction (20% to 80%) of LTS. The host defense activities examined included those of splenic and thymic cytolytic T lymphocytes (CTL) and lymphokine‐activated killer cells as well as splenic tumoricidal macrophages. Although most activities were modulated by tumor growth and/or treatment, only CTL responsiveness appeared to correlate with survival. CTL activity in the treated groups with LTS was significantly higher than in control groups late in the treatment period. Finally, ex vivo analyses of splenocytes and thymocytes together with the rejection of implanted tumor at 17 months established that LTS displayed specific long‐term immune memory. Int. J. Cancer 87:101–109, 2000.

Protective specific immunity induced by cyclophosphamide plus tumor necrosis factor α combination treatment of EL4-lymphoma-bearing C57BL/6 mice

Fig. 5 Thymic effector lytic activity — the effect of tumor rechallenge. Conditions were the same as those given in Fig. 2, except the dose of Cy used was 150 mg/kg. Mice were sacrificed on day 128 and thymocyte suspensions prepared. R mice rechallenged on day 60 by i.p. implantation of 5x 104 viable EL4 tumor cells: VR mice not rechallenged. Evaluation of cytolytic activity after stimulation culture was against both EL4 or C1498 radiolabeled tumor target cells and in both 6-h and 18-h 51 Cr-release assays. R thymocytes were also examined for cytolytic activity on day 128. Immediately after the thymus had been obtained from the mice and single-cell suspensions prepared. These fresh R thymocytes (F-R) were plated with IL-2 (2.4 ng/ml) and radiolabeled target cells and 51 Cr release was assessed 18 h later Fig. 7 Effect of combination treatment compared to CY alone on the thymic effector lytic activity. Conditions are the same as those given in Fig. 5, where the dose of CY was 150 mg/kg. Data using cells obtained on day 128 from rechallenged mice are shown

Specific anti-EL4-lymphoma immunity in mice cured 2 years earlier with doxorubicin and interleukin-2

Abstract This laboratory has reported the conditions for an effective, non-toxic, chemoimmunotherapy utilizing doxorubicin in combination with prolonged administration of interleukin-2 and the identification of the critical role of activated CD8+ T cells in the therapeutic effect. Mice (C57BL/6) cured in those studies have been followed for the remainder of their life spans. These mice, approximately 2 months of age when initially inoculated with syngeneic EL4 lymphoma, survived for more than 2 years, the normal life span of C57BL/6 mice. Mice 4 months old reinoculated with the EL4 cells all survived. At about 1 year of age mice were sacrificed and the ability of their thymocytes and splenocytes to develop specific CD8+ anti-EL4 activity was as high as it had been at the time of tumor rejection. At about 2 years of age EL4 was reimplanted into mice; all of them survived. These surviving mice, at 2 years 2 months of age, as well as a group of 2-year-old mice not rechallenged, were killed and functional antitumor activity and phenotype characteristics of various lymphocyte populations were determined in comparison to those of young and age-matched control mice. The phenotyping of the lymphocytes from the cured mice indicated very notable differences in subset distribution and increased CD44 expression. Functionally they developed high levels of anti-EL4 activity, which was ablated by combined treatment with monoclonal antibodies against CD8 and CD44, indicating the role of memory cells. Consistent with cells from aged mice, these same cell populations had a very reduced allogeneic responsiveness. It appears that cured mice have developed an immune memory specific for EL4.

Doxorubicin plus tumor necrosis factor α combination treatments in EL4- lymphoma-bearing C57BL/6 mice

Abstract The therapeutic efficacy of a total of 42 single-agent or combination protocols involving doxorubicin (Adriamycin, ADM) and tumor necrosis factor α (TNFα) were evaluated in the syngeneic murine lymphoma model, C57BL/6-EL4. Combination treatments were the most effective and the therapeutic effects were schedule-dependent; e.g. it was generally advantageous for ADM to precede TNFα administration. Two protocols selected for further study were 4 mg/kg ADM i.v. on days 1 and 8 plus TNFα, i.v., at either 16 000 U (7 μg)/injection, on days 1 and 8 or 4000 U (1.7 μg)/injection, on days 11–15. Survival of mice bearing one of four EL4 sublines having different in vitro drug sensitivities was assessed. These sublines were E10 (ADM-sensitive/TNFα-resistant), E16 (sensitive/sensitive), ER2 (ADM-resistant/TNFα-sensitive) and ER13 (resistant/resistant). Between 80% and 100% long-term survivors (i.e. tumor free on day 60) were obtained with the two treatments in mice bearing ADM-sensitive sublines, even though one of these sublines, E10, was resistant to TNFα in vitro. Induction of long-term survival appeared, therefore, to correlate with in vitro defined sensitivity/resistance to ADM, but not to TNFα. Treatment-induced modulations of tumoricidal immune effector functions were also examined. Taken together, the results indicated that induction of long-term survival involved complex interactions of: (1) ADM-induced tumor modifications, including, but not limited to, tumor debulking, (2) combination-treatment-induced modifications of splenic cytolytic T cell and macrophage activities, and (3) the restoration of thymus cellularity. Finally, when long-term survivors resulting from treatment of E10- or E16-bearing mice were implanted with ER2 on day 120, the majority survived, indicating that long-term immune memory, capable of recognizing drug resistant variants, had been established.

Thymic anti-tumor effectors in mice cured of lymphoma by cyclophosphamide plus TNF-α therapy: Phenotypic and functional characterization up to 20 months after initial tumor inoculation

As reported previously, cyclophosphamide plus tumor necrosis factor‐α treatment of C57BL/6 mice bearing advanced EL4 lymphoma induced approx. 60% long‐term (i.e., >60 days) survivors. These mice developed protective immunity, as evidenced by 1) rejection (100% survival) of EL4 tumor re‐implanted on day 60 (day 0 = initial tumor implantation); and 2) development of significant levels of specific EL4 tumor cell killing activity by both splenocytes and thymocytes. Using this model, age‐related changes in functionally and phenotypically definable thymocyte subsets were assessed. In thymocytes from 90 to 308 day survivors, specific immune memory was long term; both CD4+ and CD8+ cells were required for the ex vivo stimulation of lytic activity, but the specific anti‐EL4 cytotoxic effector was CD4−CD8+. On day 520, the surviving mice were randomized into 2 groups. One group received a second re‐challenge with EL4 tumor cells and all survived. The other group was sacrificed on day 520. Their thymocytes, exposed to X‐irradiated EL4, developed anti‐EL4 lytic activity and, in comparison with thymocytes of young and age‐matched control mice, were markedly enriched in CD4−CD8+CD44+ cells. On day 625, thymocytes from the survivors of the day 520 re‐challenge were evaluated and were found to have developed specific anti‐EL4 lytic activity. Phenotypically, they had returned toward the pattern seen in age‐matched control mice although CD4−CD8+CD44+ cells remained increased. These mice were ≥2 years old, the median life span of C57BL/6 mice. Thus, mice cured of tumor by an immuno‐modulating regimen rejected re‐implanted primary tumor and maintained specific thymic anti‐tumor immune memory for life. Int. J. Cancer 76:579–586, 1998.© 1998 Wiley‐Liss, Inc.

Polymyxin B-mediated lysis of tumor cells

Polymyxin B (PmB) in the concentration range of 10-50 micrograms/ml is used routinely in immunological studies to neutralize low levels of contaminating lipopolysaccharide (LPS) in media or reagents. While using PmB for such a purpose unexpected results were obtained, which led to the finding that low levels of PmB are cytotoxic to certain tumor cells. Further examination of a panel of 10 tumor cell lines revealed that in an 18-hour 51Cr release assay, EL4 cells and EL4/ADM cells were very sensitive (lysed by > or = 10 micrograms PmB/ml), C1498 cells and REH cells were moderately sensitive (lysed by > or = 20 micrograms PmB/ml) and cells of the remaining 6 lines were resistant (lysed only by 100 micrograms/ml) to PmB. A similar pattern of sensitivity was observed when 3H-thymidine incorporation was used as a measure of PmB effects in cell proliferation. The PmB concentration needed to kill 50% of the tumor cells in a suspension differed greatly among lines; thus for cells of a resistant line 8-fold more PmB was required for 50% killing than for those of a sensitive line. PmB toxicity toward EL4 cells was shown to increase to a plateau level with increasing time of exposure; however, the higher the concentration the earlier the plateau was reached. LPS may prevent PmB toxic effects since PmB binds to the lipid A portion of the LPS molecule, but 100 micrograms LPS/ml was only able to reduce the toxicity of 10 and 20 micrograms PmB/ml, and not that of 50 or 100 micrograms PmB/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipopolysaccharide and splenic tumoricidal macrophage activation.

Splenic macrophage tumoricidal activity was examined and a splenic macrophage tumoricidal assay was established. Initially, mixtures of lipopolysaccharide (LPS) and spleen single cell suspensions (SSCS) were cultured for 1–4 days. Adherent macrophages, washed free of nonadherent cells and LPS, were then examined and were found to lack tumoricidal activity in a standard 18‐h 51Cr release assay. However, tumoricidal activity was generated if LPS was added to the SSCS cultures at later time points during the 4‐day incubation period; maximal activity was seen when LPS was added on day 3. In parallel, significant changes in macrophage autofluorescence and morphology, but not phenotype, were observed. Next, SSCS were cultured for 1–4 days without stimulating agents. Adherent macrophages were then washed free of nonadherent cells and LPS was added. Significant tumoricidal activity developed in time‐ and LPS concentration‐dependent fashions. The presence of nonadherent spleen cells in physical contact with the macrophages during the SSCS culture was essential for the macrophages in the resultant monolayer to be responsive to LPS. Activated splenic macrophage‐mediated lysis of tumor cells was shown to depend on the contact between the two cells. J. Leukoc. Biol. 56: 714–722; 1994.

Immunoregulation by tumor necrosis factor α (TNF): an opportunity for therapeutic intervention ?

Tumor necrosis factor α (TNF), a protein cytokine, is an endogenous pleiotropic mediator of inflammatory, immune and host defense functions. It is extremely potent, acting either alone or in combination with other cytokines or factors. It acts both as an autocrine and a paracrine mediator of a wide variety of localized responses as well as in a more generalized fashion. Based on current knowledge, its critical roles as local mediator of homeostasis and host defense are considered advantageous and those on systemic metabolic responses are not. In fact, systemic TNF has been demonstrated to be a/the causative agent in septic shock, in cachexia and in certain autoimmune diseases. TNF was so named because of its extremely potent anti-tumor (hemorrhagic necrosis) activity observed following systemic administration [1]. As the understanding of the molecular and cellular mechanisms of action of this cytokine is improved, opportunities for therapeutic intervention based on this knowledge are increased and should be given further consideration.