Gintaras Zaleskis

Doxorubicin induces specific immune functions and cytokine expression in peritoneal cells

To examine the basis of the immune modulation induced by the anticancer agent doxorubicin (DOX), the immunophenotype, tumoricidal activity, cytokine protein and mRNA expression were determined using peritoneal exudate cells (PEC) from saline-treated (untreated) and DOX-treated mice. A greater percentage of PEC from DOX-treated mice than from untreated mice were adherent to plastic, had characteristics of granulocytes, and were positive for the NK1.1, CD11b/Mac-1, and CD3 markers. DOX decreased the percentage of CD45R/B220+ cells. PEC from DOX-treated mice had greater tumoricidal potential than those from untreated mice since IL2, LPS, or IFNγ alone increased the cytolytic activity of PEC from DOX-treated mice, whereas PEC from untreated mice required both LPS and IFNγ to become cytolytic. DOX treatment modulated the expression of specific cytokines. Following stimulation in culture, PEC from DOX-treated mice produced more TNF, IL1, and IFNγ than PEC from untreated mice. DOX treatment increased the levels of TNF, but not IL1, mRNA and decreased the levels of IL6 mRNA and protein. These data demonstrate that a single DOX injection induces specific effects in PEC and, as a consequence, increases the tumoricidal potential of cells of the macrophage and natural killer types.

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.

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.

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.

Effect of Perioperative Chemoimmunotherapy with Cyclophosphamide and Autologous Tumor Vaccine in Murine MBT-2 Bladder Cancer

The in vitro cytotoxic activity of splenocytes from C3H/He mice implanted subcutaneously with 10(6) syngeneic MBT-2 tumor cells on day 0 was significantly enhanced after cyclophosphamide (100 mg./kg., intraperitoneally) given 2 days before tumor resection on day 17, with or without active specific immunization with BCG plus autologous irradiated tumor cells (vaccine) 1 week after tumor resection. Furthermore, a significantly lower tumor incidence was seen in mice challenged with 10(5), but not 10(6), tumor cells per mouse 24 hours after tumor resection on day 17 and treated with cyclophosphamide on day 15 and postoperatively with vaccine than was found in nontreated tumor resected mice. Phenotypic analysis of cells from spleen showed that cyclophosphamide pretreatment and postoperative vaccine, either singly or in combination, induced a significant increase of both CD44+ memory T cells and CD11b+ myeloid/macrophage cells. Thus, in addition to a specific antitumor immune response, a nonspecific cytolytic mechanism may also play a role in the observed antitumor effect.

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.