Subtherapeutic doses of interleukin-15 augment the antitumor effect of interleukin-12 in a B16F10 melanoma model in mice.

Interleukin-12 (IL-12) is a potent immunoregulatory cytokine that exhibits antitumor activity in many experimental tumor models. In the present study, we investigated the ability of IL-15, a cytokine sharing many functions of IL-2, to modulate antitumor effectiveness of IL-12 against B16F10 melanoma in mice. In a model of locally growing tumor, intratumoral (i.t.) administration of IL-12, in three cycles of five consecutive daily injections (0.1 mug) followed by 2 days of rest, led to considerable delay of tumor development but no curative response was achieved. When combined with IL-12, subtherapeutic doses of IL-15 (0.4 mug) pontentiated the antitumor effects of IL-12 and induced complete tumor regressions in 50% of mice. Similar results were obtained in a model in which tumor-bearing mice were intravenously co-injected with melanoma cells to induce metastases. Combined administration of IL-12 and IL-15 yielded greater antitumor activity than injections of either cytokine alone and resulted in prolonged survival of mice bearing locally growing tumor and metastases. Studies of immunological parameters in mice treated with both IL-12 and IL-15 have shown enhanced NK activity (against YAC-1 cells) in the spleen and stimulation of both NK activity and specific anti-B16F10 cytotoxic effector cells in tumor-draining lymph nodes (LN). The strong antitumor effect of the IL-12 + IL-15 combination correlated with a high serum level of IFN-gamma in the treated mice. Moreover, increased expression of IL-15Ralpha was demonstrated in LN lymphocytes isolated from mice injected with IL-12. This result together with findings of other authors showing enhanced expression of IL-12 receptor by IL-15 [1] suggests that the augmentation of the antitumor effect during the course of IL-12/IL-15-based therapy could result from reciprocal upregulation of receptors by both cytokines and synergistic effects on IFN-gamma induction.     

Intratumoral immunocytokine treatment results in enhanced antitumor effects

Immunocytokines (IC), consisting of tumor-specific monoclonal antibodies fused to the immunostimulatory cytokine interleukin 2 (IL2), exert significant antitumor effects in several murine tumor models. We investigated whether intratumoral (IT) administration of IC provided enhanced antitumor effects against subcutaneous tumors. Three unique ICs (huKS-IL2, hu14.18-IL2, and GcT84.66-IL2) were administered systemically or IT to evaluate their antitumor effects against tumors expressing the appropriate IC-targeted tumor antigens. The effect of IT injection of the primary tumor on a distant tumor was also evaluated. Here, we show that IT injection of IC resulted in enhanced antitumor effects against B16-KSA melanoma, NXS2 neuroblastoma, and human M21 melanoma xenografts when compared to intravenous (IV) IC injection. Resolution of both primary and distant subcutaneous tumors and a tumor-specific memory response were demonstrated following IT treatment in immunocompetent mice bearing NXS2 tumors. The IT effect of huKS-IL2 IC was antigen-specific, enhanced compared to IL2 alone, and dose-dependent. Hu14.18-IL2 also showed greater IT effects than IL2 alone. The antitumor effect of IT IC did not always require T cells since IT IC induced antitumor effects against tumors in both SCID and nude mice. Localization studies using radiolabeled ¹¹¹In-GcT84.66-IL2 IC confirmed that IT injection resulted in a higher concentration of IC at the tumor site than IV administration. In conclusion, we suggest that IT IC is more effective than IV administration against palpable tumors. Further testing is required to determine how to potentially incorporate IT administration of IC into an antitumor regimen that optimizes local and systemic anticancer therapy.     

Differential antitumor effects of administration of recombinant IL-18 or recombinant IL-12 are mediated primarily by Fas-Fas ligand- and perforin-induced tumor apoptosis, respectively.

Systemic administration of rIL-18 protein to mice significantly suppresses the growth of murine tumor cell lines. The antitumor effect of IL-18 appears to be primarily mediated by asialo GM1+ cells. Since IL-18 enhances Fas ligand (FasL) expression on NK cell lines, the IL-18 antitumor effects could be mediated by FasL-induced cross-linking of Fas and subsequent tumor apoptosis. To address this question, rIL-18 or rIL-12 was administered to animals bearing the CL8-1 melanoma inoculated intradermally into wild type (wt), lymphoproliferation gene (lpr) (Fas deficient), or generalized lymphoproliferative disease gene (gld) (FasL deficient) mice. Although rIL-12 treatment retained significant antitumor effects in gld and lpr mice, those of rIL-18 administration were completely abrogated in gld but not lpr or wt mice. In vitro cytotoxicity was significantly enhanced against NK-sensitive YAC-1 cells and CL8-1 cells by rIL-18 administration to wt mice, but not to gld mice. Furthermore, rIL-18 administration augmented the cytotoxicity of liver lymphocytes harvested from perforin-deficient mice, whereas rIL-12 administration did not. Consistent with the role of this pathway, rIL-18 administration also up-regulates the expression of FasL mRNA in splenocytes. Lysis of CL8-1 cells induced by anti-Fas agonistic Ab was enhanced about 1.4-fold by IFN-gamma, a cytokine that is induced by IL-18 in vitro and in vivo. We conclude that the antitumor effect of IL-18 is exerted predominantly through a Fas-dependent pathway. The perforin pathway, however, appears to be the predominant cytolytic pathway mediating IL-12 antitumor effects.     

Antitumor effects of the combination immunotherapy with interleukin-12 and tumor necrosis factor α in mice

 There is strong evidence that antitumor activity of interleukin-12 (IL-12) in vivo is mediated, in part, through interferon (IFNγ) produced by IL-12-stimulated natural killer and T cells. Since IFNγ and tumor necrosis factor α (TNFα) have been reported to synergize in antitumor effects in a number of models, we decided to examine whether the combined treatment with recombinant mouse IL-12 and recombinant human TNFα would produce similar effects. The efficacy of the combined IL-12/TNFα immunotherapy was evaluated in three tumor models in mice: B16F10 melanoma, Lewis lung (LL/2) carcinoma and L1 sarcoma. Intratumoral daily injections of 1 μg IL-12 in combination with 5 μg TNFα into B16F10-melanoma-bearing mice resulted in a significant retardation of the tumor growth as compared with that in controls and in mice treated with either cytokine alone. Similar effects were obtained using 0.1 μg IL-12 and 5 μg TNFα in LL/2 carcinoma and L1 sarcoma models. Antitumor activity against L1 sarcoma was still preserved when TNFα at a low dose (1 μg) was combined with 0.1 μg IL-12 and applied for a prolonged time. Potentiation of antitumor effects, which was observed in IL-12/TNFα-based immunotherapy, could result from at least three different mechanisms, partly related to stimulation of IFNγ and TNFα production in treated mice: (a) direct cytostatic/cytotoxic effects on tumor cells, (b) induction of antitumor activity of macrophages, and (c) inhibition of blood vessel formation in the tumor. Our studies demonstrate that combination tumor immunotherapy with IL-12 and TNFα may be more effective than single-cytokine treatment, and suggest possible mechanisms by which IL-12 and TNFα may exert potentiated therapeutic effects against locally growing tumors. Received: 17 February 1997 / Accepted: 5 August 1997     

Inhibitory effect of metastasis by combined administration with interleukin-2 and sizofiran,a single glucan — immunohistochemical study —

Innovations in methods of combined administration with other BRM or chemotherapeutic drugs have been discussed. We have reported [1] that combined administration with recombinant interleukin-2 (rIL-2) and sizofiran (SPG) is effective in prolonging survival time of C57BL/6 mice intraperitoneally inoculated with EL-4 lymphoma. The immunomechanisms of the combined administration were clarified investigating the intraperitoneal cell population in the primary tumor site, especially the tumor infiltrating lymphocyte (TIL) quantitatively. In the present study, to clarify the antitumor effects of combined administration with rIL-2 and SPG on the metastatic sites, the immnunomechanisms of the suppressive effects of combined administration on the metastasis were studied in EL-4 lymphoma cells intraperitoneally transplanted to mice. Inasmuch as EL-4 lymphoma shows rapid hepatosplenic metastasis, we studied the metastatic foci in the liver and the spleen semiquantitatively investigating the histopathological and immunohistochemical findings of the metastatic foci, especially the TIL. The metastatic foci were stained by hematoxylin-eosin (HE) and monoclonal antibodies (L3T4, Lyt2, asialo GM1, Mac-1, and Ia). The combined administration resulted in: 1) fewer infiltrating tumor cells, 2) more lymphocytic infiltration, and 3) more antitumor effector cells (cytotoxic T cells: Lyt2 and natural killer cells: asialo GM1), macrophages (Mac-1), helper T cells (L3T4), and cells with MHC-class-II antigen (Ia) than did administration of rIL-2 alone or SPG alone, or no administration of these two at all. Combined administration with rIL-2 and SPG appears to activate antitumor-immune response at the metastatic site more effectively than when either agent is administered alone.     

Synergistic anti-tumor responses after administration of agonistic antibodies to CD40 and IL-2: coordination of dendritic and CD8+ cell responses

In cancer, the coordinate engagement of professional APC and Ag-specific cell-mediated effector cells may be vital for the induction of effective antitumor responses. We speculated that the enhanced differentiation and function of dendritic cells through CD40 engagement combined with IL-2 administration to stimulate T cell expansion would act coordinately to enhance the adaptive immune response against cancer. In mice bearing orthotopic metastatic renal cell carcinoma, only the combination of an agonist Ab to CD40 and IL-2, but neither agent administered alone, induced complete regression of metastatic tumor and specific immunity to subsequent rechallenge in the majority of treated mice. The combination of anti-CD40 and IL-2 resulted in significant increases in dendritic cell and CD8(+) T cell number in advanced tumor-bearing mice compared with either agent administered singly. The antitumor effects of anti-CD40 and IL-2 were found to be dependent on CD8(+) T cells, IFN-gamma, IL-12 p40, and Fas ligand. CD40 stimulation and IL-2 may therefore be of use to promote antitumor responses in advanced metastatic cancer.     

B cells are required for tumor-targeting Salmonella in host

Systemic administration of Salmonella to tumor-bearing mice leads to the preferential accumulation within tumor sites and retardation of the tumor growth. Host factors including innate and adaptive immune responses influence Salmonella-induced antitumor activity. Antitumor activities of Salmonella are not only determined by the tumor regression but also by the host immune response. Herein, we demonstrated that B cells play an important role in the antitumor activity mediated by Salmonella. Body weight and survival of B cell-deficient mice were decreased compared with wild-type, CD8(+) cell-deficient, or CD4(+) cell-deficient mice after Salmonella administration. Although Salmonella accumulated within the tumors in B cell-deficient mice, the bacterial loads of healthy organs were higher than those in wild-type mice. The inflammation cytokine and bacteremia were found in B cell-deficient mice after Salmonella treatment. When Salmonella accumulated within the tumor, B cells inhibited the dissemination of Salmonella to other healthy organs. The depletion of host B cells resulted in a noticeably higher total number of Salmonella in the tumor and inhibited tumor growth. Meanwhile, B cell-depletive and B cell-adoptive transfer of serum experiments demonstrated that the natural antibody produced by B cell takes part in the control of Salmonella dissemination in tumor-bearing mice. In this study, we want to address the mechanisms of incorporating host immunoresponse as a way to augment the antitumor activities of Salmonella.     

Sequential immunotherapy using interleukin-1 followed by interleukin-2 of ascitic MOPC104E-bearing mice

This study shows that intraperitoneal injection of interleukin-1 (IL-1), followed by interleukin-2 (IL-2), can effectively eradicate murine ascitic tumor cells. This antitumor effect of IL-1 and IL-2 was abolished when administration of IL-2 preceded that of IL-1. Solid tumors inoculated subcutaneously (s.c.) into the back of mice were also sensitive to this combined i.p. therapy, indicating a systemically-operating antitumor mechanism. Splenocytes from tumor-bearing mice treated with IL-1 followed by IL-2 showed a strong tumor-neutralizing activity. The population responsible proved to be Lyt2.2 (CD8)-positive cells.Abbreviations IL interleukin - LAK lymphokine activated killer - LU lytic unit - MST median survival time - SE sonicated tumor extract     

Antitumor and antimetastatic activity of IL-23

The structure and T cell stimulatory effects of the recently discovered cytokine IL-23 are similar to, but distinct from, those of IL-12. Although the antitumor activities of IL-12 are well characterized, the effect of IL-23 on tumor growth is not known. In this study, murine CT26 colon adenocarcinoma and B16F1 melanoma cells were engineered using retroviral vectors to release single-chain IL-23 (scIL-23) to evaluate its antitumor activity. In BALB/c mice, scIL-23-transduced CT26 cells grew progressively until day 26 to an average size of 521 +/- 333 mm(3), then the tumors started to regress in most animals, resulting in a final 70% rate of complete tumor rejection. scIL-23 transduction also significantly suppressed lung metastases of CT26 and B16F1 tumor cells. In addition, mice that rejected scIL-23-transduced tumors developed a memory response against subsequent wild-type tumor challenge. Compared with scIL-12-expressing CT26 cells, scIL-23-transduced tumors lacked the early response, but achieved comparable antitumor and antimetastatic activity. These results demonstrated that IL-23, like IL-12, provided effective protection against malignant diseases, but it probably acted by different antitumor mechanisms. As a first step in identifying these antitumor mechanisms, tumor challenge studies were performed in immunocompromised hosts and in animals selectively depleted of various lymphocyte populations. The results showed that CD8(+) T cells, but not CD4(+) T cells or NK cells, were crucial for the antitumor activity of IL-23.     

The TLR-7 agonist, imiquimod, enhances dendritic cell survival and promotes tumor antigen-specific T cell priming: relation to central nervous system antitumor immunity

Immunotherapy represents an appealing option to specifically target CNS tumors using the immune system. In this report, we tested whether adjunctive treatment with the TLR-7 agonist imiquimod could augment antitumor immune responsiveness in CNS tumor-bearing mice treated with human gp100 + tyrosine-related protein-2 melanoma-associated Ag peptide-pulsed dendritic cell (DC) vaccination. Treatment of mice with 5% imiquimod resulted in synergistic reduction in CNS tumor growth compared with melanoma-associated Ag-pulsed DC vaccination alone. Continuous imiquimod administration in CNS tumor-bearing mice, however, was associated with the appearance of robust innate immune cell infiltration and hemorrhage into the brain and the tumor. To understand the immunological mechanisms by which imiquimod augmented antitumor immunity, we tested whether imiquimod treatment enhanced DC function or the priming of tumor-specific CD8+ T cells in vivo. With bioluminescent, in vivo imaging, we determined that imiquimod dramatically enhanced both the persistence and trafficking of DCs into the draining lymph nodes after vaccination. We additionally demonstrated that imiquimod administration significantly increased the accumulation of tumor-specific CD8+ T cells in the spleen and draining lymph nodes after DC vaccination. The results suggest that imiquimod positively influences DC trafficking and the priming of tumor-specific CD8+ T cells. However, inflammatory responses induced in the brain by TLR signaling must also take into account the local microenvironment in the context of antitumor immunity to induce clinical benefit. Nevertheless, immunotherapeutic targeting of malignant CNS tumors may be enhanced by the administration of the innate immune response modifier imiquimod.     

Synergistic antitumor effects of interleukin-12 gene transfer and systemic administration of interleukin-18 in a mouse bladder cancer model

We introduced the interleukin-12 (IL-12) gene into the mouse bladder cancer cell line (MBT2) to establish sublines that secrete bioactive IL-12. IL-12-secreting MBT2 (MBT2/IL-12) sublines were completely rejected when subcutaneously implanted into immunocompetent syngeneic C3H mice. Although this antitumor effect did not change when IL-12-secreting cells were injected into immunodeficient mice whose CD8⁺ T or CD4⁺ T cells had been depleted by the corresponding antibody, it was abrogated when natural killer cells were depleted by anti-asialoGM1 antibody. In addition, when parental MBT2 cells mixed with MBT2/IL-12 cells were subcutaneously injected into mice, admixed MBT2/IL-12 inhibited the growth of the parental tumor. Furthermore, this antitumor effect was enhanced by systemic IL-18 administration. This synergism was abrogated when the mice were treated with interferon-γ-neutralizing antibody in vivo. In conclusion, local secretion of IL-12 led to effective antitumor activity that was enhanced by systemic administration of IL-18. Interferon-γ plays an important role in the synergism of IL-12 gene transduction and systemic administration of IL-18. Received: 7 May 1998 / Accepted: 27 May 1999     

Study of the effect of lithium on lymphokine-activated killer cell activity and its antitumor growth.

The in vitro effect of lithium on lymphokine-activated killer cell (LAK) activity and its in vivo antitumor growth were observed. LAK activity was enhanced when LiCl was added during LAK cell induction, and this enhancement was observed both in human peripheral blood mononuclear cell and in mouse splenocytes used as LAK precursors. Cholera toxin, which can increase intracellular levels of cAMP, decreased LAK cell activity. However, lithium partially reversed this inhibitory effect, indicating that lithium increased LAK cell activity by decreasing cAMP levels. D-Sphingosine, an inhibitor of protein kinase C, and EGTA, a calcium chelator, both inhibited the LAK cell activity. However, their inhibitory effects could not be reversed by lithium because lithium was added in the culture in combination with one of these inhibitors during LAK cell induction. By using slot blot analysis, the effect of lithium on the expression of tumor necrosis factor-alpha mRNA of LAK cells was analyzed. Lithium increased the level of tumor necrosis factor-alpha mRNA when both lithium and interleukin 2 were added to induce LAK cells. The in vivo antitumor effect of lithium has also been studied. Using a mouse melanoma experimental model, the effect of lithium on tumor growth was also observed. Both lithium alone and interleukin 2/LAK had an antitumor effect, whereas the treatment of interleukin 2/LAK in combination with lithium had the strongest inhibitory effect on tumor growth, since this treatment resulted in reduction of tumor size and prolongation of survival in tumor-bearing mice. Therefore, it is hopeful that lithium can be used as a new immunomodulator for cancer immunotherapy and immune diseases.     

Oncolytic Adenovirus Expressing IL-23 and p35 Elicits IFN-γ- and TNF-α-Co-Producing T Cell-Mediated Antitumor Immunity

Cytokine immunogene therapy is a promising strategy for cancer treatment. Interleukin (IL)-12 boosts potent antitumor immunity by inducing T helper 1 cell differentiation and stimulating cytotoxic T lymphocyte and natural killer cell cytotoxicity. IL-23 has been proposed to have similar but not overlapping functions with IL-12 in inducing Th1 cell differentiation and antitumor immunity. However, the therapeutic effects of intratumoral co-expression of IL-12 and IL-23 in a cancer model have yet to be investigated. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral inoculation of oncolytic adenovirus co-expressing IL-23 and p35, RdB/IL23/p35. Intratumoral administration of RdB/IL23/p35 elicited strong antitumor effects and increased survival in a murine B16-F10 syngeneic tumor model. The levels of IL-12, IL-23, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were elevated in RdB/IL23/p35-treated tumors. Moreover, the proportion of regulatory T cells was markedly decreased in mice treated with RdB/IL23/p35. Consistent with these data, mice injected with RdB/IL23/p35 showed massive infiltration of CD4⁺ and CD8⁺ T cells into the tumor as well as enhanced induction of tumor-specific immunity. Importantly, therapeutic mechanism of antitumor immunity mediated by RdB/IL23/p35 is associated with the generation and recruitment of IFN-γ- and TNF-α-co-producing T cells in tumor microenvironment. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-23 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.     

Combination of IL-12 gene therapy and CTX chemotherapy inhibits growth of primary B16(F10) melanoma tumors in mice

We investigated suppression of murine B16(F10) melanoma tumor growth following a therapy which involved concomitant administration of cyclophosphamide and plasmid DNA bearing interleukin-12 gene. Since both therapeutic factors display antiangiogenic capabilities, we assumed that their use in blocking the formation of new blood vessels would result in augmented inhibition of tumor growth. This combined therapy regimen indeed resulted in a considerable suppression of tumor growth. We observed a statistically significant extension of treated animals' lifespan. Interestingly, the therapeutic effect was also obtained using a plasmid without an interleukin gene insert. This observation suggests that plasmid DNA, which has been widely applied for treating neoplastic tumors, contains element(s) that elicit immune response in mice.     

Generation from tumor-bearing mice of lymphocytes with in vivo therapeutic efficacy

Systemic transfer of sensitized lymphocytes can effectively mediate the regression of established tumors. However, virtually all prior experimental applications of this approach have utilized lymphocytes from animals that have been immunized to reject tumor challenge. A similar source of cells is not available in the human. With the use of a weakly immunogenic murine tumor, MCA 105, we demonstrate here that following in vitro sensitization (IVS) with viable tumor cells and interleukin 2, the nontherapeutic lymphoid cells from mice bearing a progressively growing tumor acquired antitumor reactivity capable of mediating the regression of established pulmonary metastases. Although the IVS system induced nonspecific lymphokine-activated killer-like cytotoxic activity from lymphoid cells of normal as well as tumor-bearing mice, therapeutically active cells could only be generated from cultures initiated with lymphoid cells from tumor-bearing animals, indicating that the IVS was a secondary in vitro immune response. Without other treatment, the IVS cells could mediate antitumor effects. However, low doses of exogenous interleukin 2 administration could enhance their therapeutic efficacy. By in vivo T cell subset depletion with monoclonal antibodies, the primary effector cells were identified as belonging to cytotoxic/suppressor T cell lineage expressing the Lyt-2 phenotype. In addition, these therapeutic effector cells could be further expanded in numbers in vitro with continuous stimulation by tumor cells in the presence of interleukin 2. Compared to the number of cells initiating the culture, as many as 126 times the number of cells were obtained after 9 days of IVS followed by in vitro expansion for an additional 5 days. Studies on the kinetics of the occurrence of the pre-effector lymphocytes during tumor growth revealed that they were readily obtained from draining lymph nodes of mice with a broad range of tumor burdens as well as durations of tumor growth. The ability to generate and expand, in vitro, therapeutically active lymphocytes from tumor-bearing hosts has important implications for cellular therapy of human cancers.     

Systemic administration of IL-23 induces potent antitumor immunity primarily mediated through Th1-type response in association with the endogenously expressed IL-12

IL-23, a cytokine, which is composed of the p40 subunit shared with IL-12 and the IL-23-specific p19 subunit, has been shown to preferentially act on Th1 effector/memory CD4+ T cells and to induce their proliferation and IFN-gamma production. The IL-23 is also reported to act on Th17-CD4+ T cells, which are involved in inducing tissue injury. In this study, we examined the antitumor effects associated with systemic administration of IL-23 and their mechanisms in mouse tumor system. Systemic administration of high-dose IL-23 was achieved using in vivo electroporation of IL-23 plasmid DNA into the pretibial muscles of C57BL/6 mice. The IL-23 treatment was associated with significant suppression of the growth of pre-existing MCA205 fibrosarcoma and prolongation of the survival of treated mice without significant toxicity when compared with those of the mice treated with EGFP. Although the therapeutic outcomes were similar to those with the IL-12 treatment, the IL-23 treatment induced characteristic immune responses distinctive to those of IL-12 treatment. The IL-23 administration even at the therapeutic levels did not induce detectable IFN-gamma concentration in the serum. In vivo depletion of CD4+ T cells, CD8+ T cells, or NK cells significantly inhibited the antitumor effects of IL-23. Furthermore, the CD4+ T cells in the lymph nodes in the IL-23-treated mice showed significant IFN-gamma and IL-17 response upon anti-CD3 mAb stimulation in vitro. These results and the ones in the IFN-gamma or IL-12 gene knockout mice suggest that potent antitumor effects of IL-23 treatment could be achieved when the Th1-type response is fully promoted in the presence of endogenously expressed IL-12.     

Intratumoral hu14.18-IL-2 (IC) Induces Local and Systemic Antitumor Effects That Involve Both Activated T and NK Cells As Well As Enhanced IC Retention

hu14.18-IL-2 (IC) is an immunocytokine consisting of human IL-2 linked to hu14.18 mAb, which recognizes the GD2 disialoganglioside. Phase 2 clinical trials of i.v. hu14.18-IL-2 (i.v.-IC) in neuroblastoma and melanoma are underway and have already demonstrated activity in neuroblastoma. We showed previously that intratumoral hu14.18-IL-2 (IT-IC) results in enhanced antitumor activity in mouse models compared with i.v.-IC. The studies presented in this article were designed to determine the mechanisms involved in this enhanced activity and to support the future clinical testing of intratumoral administration of immunocytokines. Improved survival and inhibition of growth of both local and distant tumors were observed in A/J mice bearing s.c. NXS2 neuroblastomas treated with IT-IC compared with those treated with i.v.-IC or control mice. The local and systemic antitumor effects of IT-IC were inhibited by depletion of NK cells or T cells. IT-IC resulted in increased NKG2D receptors on intratumoral NKG2A/C/E(+) NKp46(+) NK cells and NKG2A/C/E(+) CD8(+) T cells compared with control mice or mice treated with i.v.-IC. NKG2D levels were augmented more in tumor-infiltrating lymphocytes compared with splenocytes, supporting the localized nature of the intratumoral changes induced by IT-IC treatment. Prolonged retention of IC at the tumor site was seen with IT-IC compared with i.v.-IC. Overall, IT-IC resulted in increased numbers of activated T and NK cells within tumors, better IC retention in the tumor, enhanced inhibition of tumor growth, and improved survival compared with i.v.-IC.     

Augmentation of antitumor effect on syngeneic murine solid tumors by an interleukin 2 slow delivery system,the IL-2 mini-pellet

We evaluated the antitumor effect of an interleukin 2 (IL-2) slow delivery system, the IL-2 mini-pellet, in two murine solid tumor models, and also investigated the enhancement of its therapeutic effect by serial administration. The IL-2 mini-pellet contains 1 × 106 units of IL-2 and releases it slowlyin vivo. In our experiment, the IL-2 mini-pellet was administered subcutaneously near the tumor site in combination with the intravenous injection of lymphokine-activated killer (LAK) cells. When this regimen was given on days 8 and 11 after the subcutaneous inoculation of Meth A fibrosarcoma into BALB/c mice, tumor growth was significantly inhibited (p < 0.05) compared to tumor growth in untreated controls. Moreover, the IL-2 mini-pellet alone was also effective in inhibiting tumor growth. In another experiment, MH134 hepatoma was inoculated into C3H/He mice. Both administration of the IL-2 minipellet alone and in combination with LAK cells resulted in complete tumor regression in four of five mice. In a third experiment, serial administration of the IL-2 mini-pellet at 3- or 5-day intervals prolonged the suppression of Meth A fibrosarcoma growth in BALB/c mice. These results suggested that the IL-2 mini-pellet could be applied to cancer immunotherapy and that its antitumor effect could be prolonged by serial administration.     

Nitric oxide short-circuits interleukin-12-mediated tumor regression

Interleukin-12 (IL-12) can promote tumor regression via activation of multiple lymphocytic and myelocytic effectors. Whereas the cytotoxic mechanisms employed by T/NK/NKT cells in IL-12-mediated tumor kill are well defined, the antitumor role of macrophage-produced cytotoxic metabolites has been more controversial. To this end, we investigated the specific role of nitric oxide (NO), a major macrophage effector molecule, in post-IL-12 tumor regression. Analysis of tumors following a single intratumoral injection of slow-release IL-12 microspheres showed an IFNγ-dependent sevenfold increase in inducible nitric oxide synthase (iNOS) expression within 48 h. Flow cytometric analysis of tumor-resident leukocytes and in vivo depletion studies identified CD11b⁺ F4/80⁺ Gr1^lo macrophages as the primary source of iNOS. Blocking of post-therapy iNOS activity with N-nitro-l-arginine methyl ester (L-NAME) dramatically enhanced tumor suppression revealing the inhibitory effect of NO on IL-12-driven antitumor immunity. Superior tumor regression in mice receiving combination treatment was associated with enhanced survival and proliferation of activated tumor-resident CD8+ T-effector/memory cells (Tem). These findings demonstrate that macrophage-produced NO negatively regulates the antitumor activity of IL-12 via its detrimental effects on CD8+ T cells and identify L-NAME as a potent adjuvant in IL-12 therapy of cancer.     

The in vivo effects of interleukin 1. I. Bone marrow cells are induced to cycle after administration of interleukin 1

We have previously reported that interleukin 1 (IL-1) administration 20 hr before irradiation protects mice from lethal effects of radiation. The recovery of total nucleated bone marrow cells and of hematopoietic progenitor cells was enhanced in IL-1 treated, as compared to untreated, irradiated mice. This suggested that IL-1 administration may affect the cells in the bone marrow of normal mice. Intraperitoneal administration of recombinant IL-1 resulted in bone marrow cell enlargement and increased cycling of these enlarged cells. In addition, the capacity of bone marrow cells from IL-1 treated mice to proliferate in response to granulocyte macrophage-colony-stimulating factor (GM-CSF) in cell suspension cultures was enhanced. The above effects were not genetically restricted as C57BL/6, B6D2F1, C3H/HeN, and C3H/HeJ mice showed similar responses. A comparative study showed that 100 ng of IL-1 was much more effective in stimulating bone marrow cells by the above criteria than 5 micrograms GM-CSF. Since IL-1, unlike CSF, can not be demonstrated to have a direct in vitro stimulatory effect on bone marrow cells, the aforementioned in vivo effects of IL-1 are presumably mediated by other hematopoietic growth factors. We have previously shown that IL-1 induces the appearance of high titers of CSF in the serum. Consequently hematopoietic growth factors that are generated at local sites following IL-1 administration may mediate the observed cell cycling effect.