Long-lasting anti-metastatic efficiency of interleukin 12-encoding plasmid DNA

Intramuscular injection of plasmid DNA encoding both subunits of the cytokine interleukin 12 (IL-12) exhibits strong antimetastatic activity against lung metastases induced by the malignant melanoma cell line B16-F10. The protective effect of IL-12 DNA is long-lasting, since administration of tumor cells 9 days after IL-12 DNA treatment prevented metastasis formation. No effects were observed with empty plasmid controls, DNA encoding the melanoma-associated antigen pmel17/gp100, the granulocyte-macrophage colony-stimulating factor GM-CSF, B7.1, or CpG-containing oligodeoxynucleotides. IL-12 DNA is required during early phases of metastasis formation and is ineffective when administered later. Its efficiency is dose dependent. The cytotoxic T cell response contributes to the antimetastatic effect as evidenced by genetically modified CD8- or perforin knockout mice. Depletion of natural killer (NK) cells by antibodies completely abrogated the effect. In contrast, the IL-12-induced antimetastatic effect was not mediated by interferon gamma (IFN-gamma) or tumor necrosis factor alpha (TNF-alpha) as shown with IFN-gamma receptor and TNF-alpha knockout mice, respectively. Toxic side effects by IL-12 were low. Our results suggest that plasmid DNA encoding IL-12 might have potential value as gene medicine against the initiation of metastasis formation.     

Antitumor and antimetastatic activity of interleukin 12 against murine tumors

It has recently been demonstrated that in vivo administration of murine interleukin 12 (IL-12) to mice results in augmentation of cytotoxic natural killer (NK)/lymphocyte-activated killer cell activity, enhancement of cytolytic T cell generation, and induction of interferon gamma secretion. In this study, the in vivo activity of murine IL-12 against a number of murine tumors has been evaluated. Experimental pulmonary metastases or subcutaneous growth of the B16F10 melanoma were markedly reduced in mice treated intraperitoneally with IL-12, resulting in an increase in survival time. The therapeutic effectiveness of IL-12 was dose dependent and treatment of subcutaneous tumors could be initiated up to 14 d after injection of tumor cells. Likewise, established experimental hepatic metastases and established subcutaneous M5076 reticulum cell sarcoma and Renca renal cell adenocarcinoma tumors were effectively treated by IL-12 at doses which resulted in no gross toxicity. Local peritumoral injection of IL-12 into established subcutaneous Renca tumors resulted in regression and complete disappearance of these tumors. IL-12 was as effective in NK cell-deficient beige mice or in mice depleted of NK cell activity by treatment with antiasialo GM1, suggesting that NK cells are not the primary cell type mediating the antitumor effects of this cytokine. However, the efficacy of IL-12 was greatly reduced in nude mice suggesting the involvement of T cells. Furthermore, depletion of CD8+ but not CD4+ T cells significantly reduced the efficacy of IL-12. These results demonstrate that IL-12 has potent in vivo antitumor and antimetastatic effects against murine tumors and demonstrate as well the critical role of CD8+ T cells in mediating the antitumor effects against subcutaneous tumors.     

Investigation of the biological effects of anti-cell adhesive synthetic peptides that inhibit experimental metastasis of B16-F10 murine melanoma cells.

The experimental metastasis of B16-F10 murine melanoma cells is blocked by the anti-cell adhesive pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS) derived from the central cell-binding domain of fibronectin. In this report, we show that peptide treatment substantially extends the survival time for mice injected intravenously with B16-F10 cells (8/8 vs. 0/8 mice alive at 150 d), thereby demonstrating the potential efficacy of GRGDS treatment in protection against metastatic colonization. We have also examined the specificity of GRGDS activity by testing a series of related homologues for their effects on experimental metastasis. The overall profile of the relative inhibitory activities of these peptides closely matched their previously established capacity to disrupt adhesion in vitro. Lung retention studies with radiolabeled B16-F10 cells revealed an accelerated rate of cell loss from the lung 0-6 h after coinjection with the active peptide GRGDS. This early effect of GRGDS was consistent with its short circulatory half-life, which was found to be 8 min. Taken together, these results suggest that peptide-mediated inhibition of pulmonary colonization is due to interference with B16-F10 cell adhesion to structures in the target organ. Possible peptide interference in tumor cell-blood cell interactions was examined in order to assess (a) possible biological side-effects of peptide treatment and (b) whether such interactions might be an alternative mechanism for GRGDS-mediated inhibition of pulmonary colonization. GRGDS was found to retain full inhibitory activity when coinjected with B16-F10 cells into mice in which platelet function was impaired by acetylsalicylic acid treatment or into thrombocytopenic mice treated with antiplatelet serum (76-93% inhibition of colony formation). These data suggest that platelet involvement in the effects of the peptide is minimal. Similarly, GRGDS was also found to be a potent inhibitor of experimental metastasis in natural killer (NK) cell-deficient beige mice (86% inhibition), thereby discounting the possibility that GRGDS artifactually enhanced NK cell activity. We conclude as a result of these studies that cell-binding fibronectin peptides are specific inhibitors of experimental metastasis that prolong survival, that they appear to function by blocking the adhesion of B16-F10 cells to structures in the target organ, and that they do not appear to act through side effects on certain metastasis-related blood cell functions. In the future, derivatives of fibronectin peptides may be potentially useful prophylactic agents for interfering with the process of metastasis.     

Combination immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation.

We examined the effectiveness of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade, alone or in combination with a granulocyte/macrophage colony-stimulating factor (GM-CSF)-expressing tumor cell vaccine, on rejection of the highly tumorigenic, poorly immunogenic murine melanoma B16-BL6. Recently established tumors could be eradicated in 80% (68/85) of the cases using combination treatment, whereas each treatment by itself showed little or no effect. Tumor rejection was dependent on CD8(+) and NK1.1(+) cells but occurred irrespective of the presence of CD4(+) T cells. Mice surviving a primary challenge rejected a secondary challenge with B16-BL6 or the parental B16-F0 line. The same treatment regimen was found to be therapeutically effective against outgrowth of preestablished B16-F10 lung metastases, inducing long-term survival. Of all mice surviving B16-BL6 or B16-F10 tumors after combination treatment, 56% (38/68) developed depigmentation, starting at the site of vaccination or challenge and in most cases progressing to distant locations. Depigmentation was found to occur in CD4-depleted mice, strongly suggesting that the effect was mediated by CTLs. This study shows that CTLA-4 blockade provides a powerful tool to enhance T cell activation and memory against a poorly immunogenic spontaneous murine tumor and that this may involve recruitment of autoreactive T cells.     

NK cells mediate Flt3 ligand-induced protection of dendritic cell precursors in vivo from the inhibition by prostate carcinoma in the murine bone marrow metastasis model

Multiple observations suggest that suppression of the dendritic cell (DC) system might be one of the mechanisms used by the tumors to escape immune response. However, no in vivo data are available to support these in vitro observations. Here we have shown that murine prostate cancer inhibits DC generation (dendropoiesis) from the bone marrow precursors in the in vivo model in mice injected intrafemorally with RM1 prostate adenocarcinoma cells. Phenotyping of DC, generated from in vivo RM1-treated bone marrow cells, revealed a significant inhibition of dendropoiesis assessed as a percentage of CD11c+MHCII+, CD11c+CD86+, and CD11c+CD80+ cells. The stimulatory capacity of these DCs to induce T cell proliferation was also markedly decreased. Notably, Flt3 ligand-based therapy reversed the inhibitory effects of prostate cancer on dendropoiesis in vivo in wild-type and C.B-17 SCID (T and B cell deficient) mice, but not in SCID beige (T, B, and NK cell deficient) animals, suggesting a key role of NK cells in Flt3 ligand-mediated protection of dendropoiesis from tumor-induced inhibition in vivo. Thus, these data demonstrate that prostate cancer inhibits DC hematopoietic precursors in vivo in the bone marrow and this effect could be abolished by a systemic administration of growth factor Flt3 ligand.     

Oncolytic adenovirus co-expressing IL-12 and IL-18 improves tumor-specific immunity via differentiation of T cells expressing IL-12Rβ2 or IL-18Rα

The oncolytic adenovirus (Ad) is currently being advanced as a promising antitumor remedy as it selectively replicates in tumor cells and can transfer and amplify therapeutic genes. Interleukin (IL)-12 induces a potent antitumor effect by promoting natural killer (NK) cell and cytotoxic T cell activities. IL-18 also augments cytotoxicity of NK cells and proliferation of T cells. This effect further enhances the function of IL-12 in a synergistic manner. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral administration of oncolytic Ad co-expressing IL-12 and IL-18, RdB/IL-12/IL-18. Intratumoral administration of RdB/IL-12/IL-18 improved antitumor effects, as well as increased survival, in B16-F10 murine melanoma model. The ratio of T-helper type 1/2 cytokine as well as the levels of IL-12, IL-18, interferon-γ and granulocyte-macrophage colony-stimulating factor was markedly elevated in RdB/IL-12/IL-18-treated tumors. Mice injected with RdB/IL-12/IL-18 also showed enhanced cytotoxicity of tumor-specific immune cells. Consistent with these results, immense necrosis and infiltration of NK cells, as well as CD4+ and CD8+ T cells, were observed in RdB/IL-12/IL-18-treated tumor tissues. Importantly, tumors treated with RdB/IL-12/IL-18 showed an elevated number of T cells expressing IL-12Rβ2 or IL-18Rα. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-18 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.     

Marrow stromal cells for interleukin-2 delivery in cancer immunotherapy

Marrow stromal cells (MSCs) can be easily gene-modified and clonally expanded making them ideal candidates for transgenic cell therapy. However, recent reports suggest that MSCs possess immunosuppressive effects, which may limit their clinical applications. We investigated whether interleukin (IL)-2 gene-modified MSCs can be used to mount an effective immune response against the poorly immunogenic B16 melanoma model. We first show that primary MSCs mixed with B16 cells and injected subcutaneously in syngeneic recipients do not affect tumor growth. On the other hand, IL-2-producing MSCs mixed with B16 cells significantly delayed tumor growth in an IL-2 dose-dependent manner. Furthermore, we observed that matrix-embedded IL-2-producing MSCs injected in the vicinity of preestablished B16 tumors led to absence of tumor growth in 90% of treated mice (p < 0.001). We demonstrated that tumor-bearing mice treated with IL-2-producing MSCs developed CD8-mediated tumor-specific immunity and significantly delayed tumor growth of a B16 cell challenge (p < 0.05). In addition, treatment of cd8-/-, cd4-/- and beige mice revealed that CD8+ and natural killer (NK) cells, but not CD4+ cells, were required to achieve antitumor effect. In conclusion, MSCs can be exploited to deliver IL-2 and generate effective immune responses against melanoma in mice with normal immune systems.     

Strengthening of antitumor immune memory and prevention of thymic atrophy mediated by adenovirus expressing IL-12 and GM-CSF

Interleukin (IL)-12 and granulocyte-monocyte colony-stimulating factor (GM-CSF) have recently been used as immunotherapeutic agents in cancer gene therapy. IL-12 and GM-CSF have differential roles in the antitumor immune response, as IL-12 targets T, NK and natural killer T (NKT) cells and GM-CSF principally targets antigen-presenting cells (APCs). To strengthen the therapeutic efficacy of these two cytokines, we generated an oncolytic adenovirus (Ad), Ad-ΔB7/IL12/GMCSF, coexpressing IL-12 and GM-CSF. Using a murine B16-F10 syngeneic tumor model, we show that Ad-ΔB7/IL12/GMCSF promoted antitumor responses and increased survival compared with an oncolytic Ad expressing IL-12 or GM-CSF alone (Ad-ΔB7/IL12 or Ad-ΔB7/GMCSF, respectively). By measuring cytotoxic T lymphocyte activity and interferon-γ production, we show that the enhanced therapeutic effect was mediated by the induction of immune cell cytotoxicity. In situ delivery of Ad-ΔB7/IL12/GMCSF resulted in massive infiltration of CD4(+) T cells, CD8(+) T cells, NK cells and CD86(+) APCs into the tissue surrounding the necrotic area of the tumor. Moreover, GM-CSF effectively promoted antitumor immune memory, which was significantly augmented by IL-12. Lastly, IL12-expressing oncolytic Ads prevented tumor-induced thymic atrophy and was associated with reduced apoptosis and increased proliferation in the thymus. Taken together, these data demonstrate that an oncolytic Ad coexpressing IL-12 and GM-CSF is a potential therapeutic tool for the treatment of cancer.     

Reinforcing B16F10/GPI-IL-21 vaccine efficacy against melanoma by injecting mice with shZEB1 plasmid or miR200c agomir

In this study, we hypothesized that the inhibition of epithelial to mesenchymal transition (EMT) program by knockdown of Zinc-finger E-box binding homeobox 1 (ZEB1) or administration of miR200c agomir would strengthen the B16F10 cells transfected with GPI-anchored IL-21 (B16F10/GPI-IL-21) vaccine efficacy in inhibiting the melanoma metastasis. Our findings from the current study indicated that, when compared with the mice immunized with the B16F10/GPI-IL-21 vaccine alone, the mice immunized with B16F10/GPI-IL-21 vaccine combined with injection of shZEB1 plasmid or miR200c agomir not only meaningfully inhibited EMT of melanoma, reduced the EMT characteristic molecular expression in tumor tissues, but also significantly decreased the Treg cells and TGF-β1, enhanced the cytotoxicities of NK cells and cytotoxic T lymphocytes and the IFN-γ level. Furthermore, the immunotherapeutic combination resulted in inhibiting the melanoma growth and lung metastasis. Our study demonstrated that using the B16F10/GPI-IL-21 vaccine in combination with the down-regulated ZEB1 or miR200c administration effectively elicited anti-tumor immunity and reduced melanoma metastasis by inhibiting the EMT program in the B16F10 melanoma-bearing mice.     

Presence of cytotoxic B220+CD3+CD4-CD8- cells correlates with the therapeutic efficacy of lymphoma treatment with IL-2 and/or IL-12

Cancer treatment with IL-2 and IL-12 is thought to work via enhancement of proliferation and activity of T cells and NK cells. Incubation of cytotoxic T lymphocytes (CTLs) and NK cells with IL-2 and/or IL-12 results in propagation of a distinct cell type called lymphokine-activated killers (LAK) characterized by increased lytic activity against many tumor types. Here we address the question whether cytokine therapy may be efficient in treatment of a LAK-insensitive tumor and, if so, which cell type, other than classic LAK cells, is responsible for tumor cell killing. We used DBA/2 mice bearing metastasized SL2 lymphoma and treated them with locally applied IL-2 and /or IL-12 injections. We showed that IL-12 treatment is efficient, though there is a rather narrow range of effective doses because of toxicity. This toxicity may be alleviated by a single injection of IL-12 before treatment. Next, we showed that IL-12 synergistically enhances the efficacy of local IL-2 treatment. Moreover, our results indicate that the IL-2/IL-12-mediated therapeutic effect is greatest when it is given after establishment of an immune response to a tumor. Finally, we showed the existence of a unique population of lymphoid cells, namely B220+CD3+CD4-CD8-, at the site of tumor growth. These cells become highly cytotoxic to SL2 cells in mice treated with cytokines late (day 10-14) in the course of the immune response, but not in mice treated early (day 3-7), and cytotoxicity of this unique cell population correlates with the success of therapy.     

c-kit+ stem cells and thymocyte precursors in the livers of adult mice

Livers of the adult mice contain c-kit+ stem cells that can reconstitute thymocytes, multiple lineage cells, and bone marrow (BM) stem cells. Transfer of 1 x 10(7) hepatic mononuclear cells (MNC) and 5 x 10(4) hepatic c-kit+ cells of BALB/c mice induced DP thymocytes within a week in four Gy-irradiated CB17/-SCID mice, but 2 wk were required for BM cells or BM c-kit+ cells to produce DP thymocytes. Moreover, B cell-depleted BM cells or liver MNC of SCID mice that had been rescued by hepatic MNC of BALB/c mice again reconstituted thymus and B cells of other irradiated SCID mice. CD3- IL-2R beta- populations of both BM cells and hepatic MNC of C57BL/6 (B6) mice could generate T cells with intermediate TCR (mostly NK1.1-) in the liver of irradiated B6 SCID mice before thymic reconstitution (extrathymic T cells). Furthermore, transfer of liver c-kit+ cells of B6-Ly 5.1 mice into irradiated B6 SCID (Ly5.2) mice revealed that liver c-kit+ cells can reconstitute myeloid and erythroid lineage cells. These results strongly suggest that the liver contains pluripotent stem cells and serves an important hematopoietic organ even into adulthood.     

Suppression of tumor formation in lymph nodes by L-selectin-mediated natural killer cell recruitment

Natural killer (NK) cells are known to reject certain tumors in vivo; however, the ability of NK cells to prevent metastasis of tumors into secondary lymphoid organs has not been addressed. Here, we report that in tumor-bearing hosts, NK cells are recruited to regional lymph nodes in wild-type mice, but not in mice deficient for L-selectin or L-selectin ligands. By adoptive transfer and complete Freund's adjuvant stimulation experiments, we demonstrated that L-selectin on NK cells and L-selectin ligands on endothelial cells are essential for NK cell recruitment to lymph nodes. Furthermore, freshly isolated resident lymph node NK cells lysed tumors efficiently, and metastasis of B16 melanoma cells to draining lymph nodes was suppressed in wild-type or Rag-1-deficient mice, but not when NK cells were depleted. Although L-selectin-deficient NK cells efficiently lysed tumor cells in vitro, NK cell-dependent suppression of tumor metastasis was diminished in mice deficient for L-selectin or L-selectin ligands because of insufficient NK cell recruitment to lymph nodes. Moreover, tumor metastasis was substantially inhibited in L-selectin-deficient mice reconstituted with wild-type NK cells. These findings indicate that L-selectin-mediated NK cell recruitment plays a crucial role in the control of tumor metastasis into secondary lymphoid organs.     

Thymic selection of H-2-incompatible bone marrow cells in SCID mice. Differences in T help for induction of B cell IgG responses versus cytotoxic T cells

Mice with congenital severe combined immunodeficiency disease (SCID) failed to mount either a T cell-independent IgM or T cell-dependent IgG anti-vesicular stomatitis virus (VSV) Indiana (IND) response. They did not generate cytotoxic T cells against lymphocytic choriomeningitis virus (LCMV) or vaccinia virus, but exhibited NK cell-like activities. When SCID mice were given bone marrow from syngeneic BALB/c (H-2d) nu/nu mice, all immune responses were expressed at control levels. If SCID mice were reconstituted with allogeneic H-2b C57BL/6 nu/nu bone marrow, the following primary anti-viral immune responses were measured. T-independent IgM anti-VSV-IND were normal, but T-dependent IgG anti-VSV-IND responses were absent. Cytotoxic T cell responses to LCMV and vaccinia virus were within normal ranges, were donor cell mediated, and were specific exclusively for the recipient SCID H-2d type. Since antigen presentation by spleen cells was functional in these chimaeras, the presented results indicate that (a) thymic selection of T cell restriction is strict; and (b) the type of T help necessary for B cells depends upon H-2-restricted contact between T and B cells, whereas, such contact-dependent help is not mandatory for the induction of virus-specific cytotoxic T cells.     

Intracranial paracrine interleukin-2 therapy stimulates prolonged antitumor immunity that extends outside the central nervous system

To explore the potential efficacy of local cytokine delivery against tumors in the central nervous system (CNS), C57BL6 mice were simultaneously given intracranial injections of tumor challenge and of irradiated B16F10 melanoma cells transduced to secrete interleukin-2 (IL-2). Intracranial IL-2 therapy generated antitumor responses capable of extending the survival of animals that received simultaneous intracranial tumor challenge either locally or at distant sites in the brain. Nontransduced melanoma cells had little effect. Animals that survived intracranial IL-2 therapy and tumor challenge showed prolonged survival compared with controls when challenged with a second tumor dose 70 days after initial treatment. In addition, animals that rejected intracranial tumors were also protected from tumor growth upon rechallenge at sites outside the CNS (i.e., subcutaneous tumor challenge). Conversely, identical or 10-fold larger doses of IL-2-transduced cells administered by subcutaneous injection failed to generate protection against intracranial tumor challenges. Elimination of T-cell and natural killer (NK) subsets using gene knockout mice and antibody-depletion techniques demonstrated that NK cells were most important for the initial antitumor response, whereas CD4+ T-cells were not necessary. These studies demonstrate that local IL-2 therapy in the brain not only generates an immediate local antitumor immune response, but also establishes long-term immunologic memory capable of eliminating subsequent tumor challenges within and outside of the CNS. Furthermore, the antitumor response to paracrine IL-2 in the brain differed significantly from that in the flank, suggesting that the intrinsic CNS cells involved in initiating immunity within the brain have different cytokine requirements from their peripheral counterparts.     

The effects of mesenchymal stem cells injected via different routes on modified IL-12-mediated antitumor activity

Owing to its tumor tropism and prolonged transgene expression, mesenchymal stem cell (MSC) has been considered as an ideal delivery vehicle for cancer gene therapies or therapeutic vaccines. In this study, we demonstrated that intratumoral (i.t.) injection of MSCs expressing modified interleukin-12 (MSCs/IL-12M) exhibited stronger tumor-specific T-cell responses and antitumor effects as well as more sustained expressions of IL-12 and interferon (IFN)-γ in both sera and tumor sites than did IL-12M-expressing adenovirus (rAd/IL-12M) in mice bearing both solid and metastatic tumors. Subcutaneous (s.c.) injection of MSCs/IL-12M at contralateral site of tumor exhibited similar levels of serum IL-12 and IFN-γ as i.t. injection, but much weaker antitumor effects in both B16F10 melanoma and TC-1 cervical cancer models than i.t. injection. Although intravenous (i.v.) injection elicited earlier peak serum levels of cytokines, it induced weaker tumor-specific T-cell responses and antitumor effects than i.t. injection, indicating that serum cytokine levels are not surrogate indicators of antitumor effects. Taken together, these results indicated that MSC is more efficient than adenovirus as a cytokine gene delivery vehicle and that i.t. injection of MSCs/IL-12M is the best approach to induce strong tumor-specific T-cell responses that correlate with anti-metastatic effects as well as inhibition of solid tumor growth, although MSCs themselves have an ability to migrate into the tumor site. In addition, MSCs/IL-12M embedded in Matrigel (MSCs/IL-12M/Matrigel) exhibited significant antitumor effects even in immunodeficient mice such as SCID and BNX mice lacking T, B and natural killer (NK) cells, but not in IFN-γ knockout mice. Our findings provide an optimal approach for designing an efficient clinical protocol of MSC-based cytokine gene therapy to induce strong tumor-specific T-cell responses and therapeutic anticancer efficacy.     

Innate immune surveillance of spontaneous B cell lymphomas by natural killer cells and gammadelta T cells

Few studies have demonstrated that innate lymphocytes play a major role in preventing spontaneous tumor formation. We evaluated the development of spontaneous tumors in mice lacking beta-2 microglobulin (beta2m; and thus MHC class I, CD1d, and CD16) and/or perforin, since these tumor cells would be expected to activate innate effector cells. Approximately half the cohort of perforin gene-targeted mice succumbed to spontaneous disseminated B cell lymphomas and in mice that also lacked beta2m, the lymphomas developed earlier (by more than 100 d) and with greater incidence (84%). B cell lymphomas from perforin/beta2m gene-targeted mice effectively primed cell-mediated cytotoxicity and perforin, but not IFN-gamma, IL-12, or IL-18, was absolutely essential for tumor rejection. Activated NK1.1+ and gammadeltaTCR+ T cells were abundant at the tumor site, and transplanted tumors were strongly rejected by either, or both, of these cell types. Blockade of a number of different known costimulatory pathways failed to prevent tumor rejection. These results reflect a critical role for NK cells and gammadeltaTCR+ T cells in innate immune surveillance of B cell lymphomas, mediated by as yet undetermined pathway(s) of tumor recognition.     

Abnormal Development of Intestinal Intraepithelial Lymphocytes and Peripheral Natural Killer Cells in Mice Lacking the IL-2 Receptor β Chain

The interleukin-2 receptor β chain (IL-2Rβ) is expressed on a variety of hematopoietic cell types, including natural killer (NK) cells and nonconventional T lymphocyte subsets such as intestinal intraepithelial lymphocytes (IEL). However, the importance of IL-2Rβ-mediated signaling in the growth and development of these cells has yet to be clearly established. We have investigated IEL and NK cells in mice deficient for IL-2Rβ and describe here striking defects in the development of these cells. IL-2Rβ^−/− mice exhibited an abnormal IEL cell population, characterized by a dramatic reduction in T cell receptor αβ CD8αα and T cell receptor γδ lymphocytes. This selective decrease indicates that IEL can be classified into those whose development and/or differentiation is dependent on IL-2Rβ function and those for which IL-2Rβ–mediated signaling is not essential. NK cell development was also found to be disrupted in IL-2Rβ–deficient mice, characterized by a reduction in NK1.1⁺CD3⁻ cells in the peripheral circulation and an absence of NK cytotoxic activity in vitro. The dependence of NK cells and certain subclasses of IEL cells on IL-2Rβ expression points to an essential role for signaling through this receptor, presumably by IL-2 and/or IL-15, in the development of lymphocyte subsets of extrathymic origin.The cytokine interleukin-2 (IL-2) and its receptor (IL2R) have long been known to play a role of prime importance in the activation and proliferation of T lymphocytes (reviewed in reference 1). However, it is unclear whether signaling via the IL-2R is required for the normal development and differentiation of other lymphoid or myeloid cells. The IL-2R is expressed on T lymphocytes (1), B cells (2), NK cells (3), neutrophils (4), and monocytes (5). The high affinity IL-2R is composed of three subunits, the α, β, and γ chains, whereas the intermediate affinity form contains only the β and γ chains (6, 7). Components of the IL-2R have been identified in other cytokine receptors: the IL-2Rγ chain (γ_c, or common γ chain) is common to the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors, whereas the IL-2Rβ chain is shared between the IL-2 and IL-15 receptors (8).The importance of individual IL-2R chains in signal transduction has been clarified recently by the engineered deletion of the gene product for each of the three known receptor subunits. Given the diverse nature of the cytokine receptors that contain the β and γ chains, and the wide range of cell types that express components of the IL-2R, the disruption of multiple immunomodulatory roles might be expected in mice deficient for any one of the three subunits. Mice deficient in IL-2Rα exhibit polyclonal T and B cell expansion (9), which correlates with a defect in activation-induced cell death in T cells, and the development of autoimmune disorders and inflammatory bowel disease. Similarly, in IL-2Rβ–deficient mice, T cells are spontaneously activated, resulting in plasma cell accumulation and high levels of autoantibodies (10). IL-2Rγ–deficient mice also show a defect in mature T and B cell development, but in contrast with the lymphoproliferation seen in IL-2Rα– and IL-2Rβ–deficient mice, a 10-fold reduction in absolute lymphocyte numbers is observed (11). In addition, NK cells are completely absent in IL-2Rγ–deficient mice, and the number of intestinal intraepithelial cells (IEL)¹ cells is severely diminished (11). In humans, loss of the γ_c function leads to X-linked severe combined immunodeficiency (SCID) (12), a disease characterized by multiple defects in T, NK, and B cell development, as might be expected from the impairment of several cytokine receptors (13). Thus, signaling via components of the IL-2R plays an essential regulatory role in homeostasis and autoimmunity.The importance of IL-2 and IL-15 in NK cell activation and proliferation has been well documented (1, 8, 14). Because both cytokines signal via receptors containing the β and γ chains, and because IL-2Rγ–deficient mice exhibit profound defects in IEL and NK cell development, it was of interest to examine the role of IL-2Rβ in these lymphocyte populations. We report here that expression of IL-2Rβ is crucial for the normal development of both TCRγδ and TCRαβ CD8αα IEL and for NK cells. These results suggest that certain cell populations that undergo extrathymic development and differentiation have an important developmental requirement for either IL-15 and/or IL-2.     

Microbial translocation augments the function of adoptively transferred self/tumor-specific CD8+ T cells via TLR4 signaling

Lymphodepletion with total body irradiation (TBI) increases the efficacy of adoptively transferred tumor-specific CD8(+) T cells by depleting inhibitory lymphocytes and increasing homeostatic cytokine levels. We found that TBI augmented the function of adoptively transferred CD8(+) T cells in mice genetically deficient in all lymphocytes, indicating the existence of another TBI mechanism of action. Additional investigation revealed commensal gut microflora in the mesenteric lymph nodes and elevated LPS levels in the sera of irradiated mice. These findings correlated with increased dendritic cell activation and heightened levels of systemic inflammatory cytokines. Reduction of host microflora using antibiotics, neutralization of serum LPS using polymyxin B, or removal of LPS signaling components using mice genetically deficient in CD14 and TLR4 reduced the beneficial effects of TBI on tumor regression. Conversely, administration of microbial ligand-containing serum or ultrapure LPS from irradiated animals to nonirradiated antibody-lymphodepleted mice enhanced CD8(+) T cell activation and improved tumor regression. Administration of ultrapure LPS to irradiated animals further enhanced the number and function of the adoptively transferred cells, leading to long-term cure of mice with large B16F10 tumors and enhanced autoimmune vitiligo. Thus, disruption of the homeostatic balance between the host and microbes can enhance cell-based tumor immunotherapy.     

K562-DC融合瘤苗刺激的CIK/NK细胞在荷瘤NOD/SCID小鼠体内的杀瘤活性

目的 探讨经K562细胞-树突细胞(DC)融合瘤苗刺激的脐血源性细胞因子诱导的杀伤(CIK)/自然杀伤(NK)细胞在荷瘤NOD/SCID小鼠体内的杀伤效能及其安全性.方法 通过不同细胞因子的组合分别诱导脐血单个核细胞(MNC)成为DC和CIK/NK细胞,通过聚乙二醇(PEG)将灭活K562细胞与DC融合形成K562-DC融合瘤苗.以10:1比例在CIK/NK细胞培养体系中加入K562-DC融合瘤苗,制备K562-DC融合瘤苗刺激的CIK/NK细胞.小鼠均经尾静脉接种K562细胞1×106诱导荷瘤鼠;于接种肿瘤细胞后24 h按不同分组分别经尾静脉输注K562-DC融合瘤苗刺激的CIK/NK细胞1×107、CIK/NK细胞1×107,同时设相应的非荷瘤鼠对照.比较各组小鼠的生存率、存活时间;用流式细胞术检测小鼠外周血、肝、肺组织中人CD13+细胞和外周血人CD56+细胞.结果 在接种1×106K562细胞后未经处理的小鼠39 d内全部死亡,8只鼠中肉眼可见瘤块的小鼠5只,其中位于肝脏的4只、脾脏的1只.接种K562细胞后接受K562-DC瘤苗刺激的CIK/NK细胞治疗的8只小鼠第65天时死亡1只,抗瘤有效率为87.5%;接受CIK/NK细胞治疗小鼠死亡2只,时间分别为接种K562细胞后第56天及第62天,抗瘤有效率为75.0%,均未发现肉眼可见瘤块.该两组小鼠存活时间分别为(69.4±1.8)d、(67.2±5.3)d,两组间差异无统计学意义(P＞0.05),均高于未予治疗的荷瘤对照组[(30.4±4.6)d](P＜0.01),荷瘤后接受K562-DC瘤苗刺激的和未经瘤苗刺激的CIK/NK细胞治疗的两组其余小鼠存活均超过70 d.经CIK/NK细胞治疗的两组存活小鼠外周血,肝、肺组织中人CD13+细胞率差异无统计学意义,均显著低于荷瘤未治疗组(P＜0.01),而治疗的两组存活小鼠外周血人CD56+细胞率与输注不同CIK/NK细胞的非荷瘤对照组相比差异无统计学意义(P＞0.05).结论 经过灭活肿瘤细胞预孵育的脐血源性CIK/NK细胞接种在动物体内具有抗瘤活性,无致瘤性.     

Activation of natural killer T cells potentiates or prevents experimental autoimmune encephalomyelitis.

Natural killer (NK) T cells recognize lipid antigens in the context of the major histocompatibility complex (MHC) class 1-like molecule CD1 and rapidly secrete large amounts of the cytokines interferon (IFN)-gamma and interleukin (IL)-4 upon T cell receptor (TCR) engagement. We have asked whether NK T cell activation influences adaptive T cell responses to myelin antigens and their ability to cause experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. While simultaneous activation of NK T cells with the glycolipid alpha-galactosylceramide (alpha-GalCer) and myelin-reactive T cells potentiates EAE in B10.PL mice, prior activation of NK T cells protects against disease. Exacerbation of EAE is mediated by an enhanced T helper type 1 (Th1) response to myelin basic protein and is lost in mice deficient in IFN-gamma. Protection is mediated by immune deviation of the anti-myelin basic protein (MBP) response and is dependent upon the secretion of IL-4. The modulatory effect of alpha-GalCer requires the CD1d antigen presentation pathway and is dependent upon the nature of the NK T cell response in B10.PL or C57BL/6 mice. Because CD1 molecules are nonpolymorphic and remarkably conserved among different species, modulation of NK T cell activation represents a target for intervention in T cell-mediated autoimmune diseases.