Successful adoptive immunotherapy of murine poorly immunogenic tumor with specific effector cells generated from gene-modified tumor-primed lymph node cells

We previously reported that cytokine gene transfer into weakly immunogenic tumor cells could enhance the generation of precursor cells of tumor-reactive T cells and subsequently augment antitumor efficacy of adoptive immunotherapy. We investigated whether such potent antitumor effector T cells could be generated from mice bearing poorly immunogenic tumors. In contrast to similarly modified weakly immunogenic tumors, MCA102 cells, which are chemically induced poorly immunogenic fibrosarcoma cells transfected with cDNA for IL-2, IL-4, IL-6, IFN-gamma, failed to augment the host immune reaction. Because priming of antitumor effector T cells in vivo requires two important signals provided by tumor-associated Ags and costimulatory molecules, these tumor cells were cotransfected with a B7-1 cDNA. Transfection of both IFN-gamma and B7-1 (MCA102/B7-1/IFN-gamma) resulted in regression of s.c. tumors, while tumor transfected with other combinations of cytokine and B7-1 showed progressive growth. Cotransfection of IFN-gamma and B7-1 into other poorly immunogenic tumor B16 and LLC cells also resulted in the regression of s.c. tumors. Cells derived from lymph nodes draining MCA102/B7-1/IFN-gamma tumors showed potent antitumor efficacy, eradicating established pulmonary metastases, but this effect was not seen with parental tumors. This mechanism of enhanced antitumor efficacy was further investigated, and T cells with down-regulated L-selectin expression, which constituted all the in vivo antitumor reactivity, were significantly increased in lymph nodes draining MCA102/B7-1/IFN-gamma tumors. These T cells developed into potent antitumor effector cells after in vitro activation with anti-CD3/IL-2. The strategy presented here may provide a basis for developing potent immunotherapy for human cancers.     

The immunological basis of tumor rejection: the absolute dependence of the effector arm on sensitized T cells after chemoimmunotherapy of a murine sarcoma

The mechanisms of tumor rejection were investigated by using a therapeutic model system involving treatment of C57BL/6J (B6) mice bearing the syngeneic MCA/76-9 or the unrelated MCA/76-64 sarcomas with cytoxan and tumor-sensitized T lymphocytes. Separated tumor-associated T lymphocytes (TAL) and tumor-associated macrophages (TAM) isolated from the regressing tumors 8 to 10 days after combination therapy expressed relatively specific cytotoxicity in vitro, whereas the unseparated tumor-associated cells (TAC), consisting of a mixture of TAL and TAM, expressed nonspecific cytotoxicity. TAM-mediated cytotoxicity was not dependent on the presence of TAL, as shown by T cell depletion of TAM or TAC cultures with the use of monoclonal anti-Thy-1 or anti-Lyt-2 antibody and complement. In contrast, the nonspecific cytotoxicity was dependent on the presence of T cells. In vivo assays using the Winn test failed to confirm certain aspects of the in vitro data. Without exception, the TAC inhibited tumor growth in an immunologically specific manner, having no effect on the growth of the unrelated B6 sarcoma. T cell depletion completely abrogated in vivo cytotoxicity. Specificity of tumor growth inhibition was confirmed in a bystander experiment in which TAC were mixed with both tumor cell types and were injected into recipient B6 mice. Tumors grew under these conditions, but the tumor that grew consisted only of those tumor cells toward which TAC cytotoxicity was not specifically directed. A bioassay indicated that the specifically immune antitumor effects at the site of regression were initiated between days 3 and 7 after combination therapy. By days 7 and 9, few tumorigenic stem cells could be detected at the tumor site. However, T cell depletion of the TAC isolated on days 8 to 10 resulted in enhanced tumor growth when the depleted TAC were injected into recipient mice. The conclusions reached were that tumor rejection was absolutely dependent on T cell participation at the tumor site, and that if TAM were involved, they required the presence of TAL and did not express nonspecific antitumor cytotoxicity. Indeed, the accelerated tumor growth seen in the absence of TAL suggested the possibility that TAM were growth stimulatory.     

siRNA干扰MMP-9和FAK双基因抑制小鼠黑色素瘤生长和在体迁移

目的:观察干扰MMP-9和FAK双基因对恶性黑色素瘤高转移细胞B16F10体内转移的影响。方法:构建PGV102-MMP9-siRNA、PGV102-FAK-siRNA重组质粒载体,脂质体^TM2000介导转染小鼠黑色素瘤B16F10细胞,RT-PCR检测基因的干扰效果;建立C57BL/6小鼠皮下移植瘤模型观察细胞在体成瘤和肿瘤的生长情况,常规组织切片,H&E染色观察肿瘤组织病理学特征;经C57BL/6小鼠尾静脉注射细胞5×10⁵个/只,24天后计数小鼠肺转移结节数评价肿瘤细胞在体迁移能力。结果:RT-PCR结果表明,重组质粒转染细胞组的MMP-9和FAK的mRNA水平显著低于正常细胞组(P<0.01),转染细胞组C57BL/6小鼠皮下成瘤的肿瘤生长速率、黑色素瘤肺转移结节数明显低于正常细胞组(P<0.01)。结论:干扰B16F10细胞MMP-9和FAK双基因可明显抑制小鼠体内恶性肿瘤的生长和迁移。     

Suppression of in vivo tumor growth by the transfection of the interleukin-5 gene into colon tumor cells

To investigate the influence of tumor producing interleukin-5 (IL-5) on growth kinetics of tumors, we transduced the murine IL-5 gene into murine colon C26 tumor cells. Two IL-5-secreting clones, low-level IL-5 producer C26-8B and high-level IL-5 producer C26-6F, were established. Both tumors, C26-6F and C26-8B, grew more slowly than the mock C26 tumor, although the in vitro growth rate of these IL-5 transfectants was much the same as that of the mock C26 cells. There was a significantly decreased number of colonies in the lung of mice given C26-6F or C26-8B tumors i.v. than in mice given mock C26 tumors i.v. Moreover, in mice given C26-6F cells i.v., a smaller number of tumor colonies in the lung was observed, as compared to the case with C26-6B cells. While the growth rate of C26-8B tumors in mice treated with anti-IL-5 mAb was more rapid than that seen in control mAb-treated mice, growth of C26-6F tumors in anti-IL-5-mAb-treated mice was slightly more rapid compared to findings in control mAb-treated mice. The isotypematched mAb did not alter the in vitro growth of mock-C26 cells or of the IL-5-gene-modified C26 cells. Growth of IL-5-secreting C26 tumors transplanted in nude mice was also inhibited. These results suggest that tumor-producing IL-5 inhibits growth of colon tumors mediated through T-cell-independent protective mechanisms of the host.     

Suppression of in vivo tumor growth by the transfection of the interleukin-5 gene into colon tumor cells

To investigate the influence of tumor producing interleukin-5 (IL-5) on growth kinetics of tumors, we transduced the murine IL-5 gene into murine colon C26 tumor cells. Two IL-5-secreting clones, low-level IL-5 producer C26-8B and high-level IL-5 producer C26-6F, were established. Both tumors, C26-6F and C26-8B, grew more slowly than the mock C26 tumor, although the in vitro growth rate of these IL-5 transfectants was much the same as that of the mock C26 cells. There was a significantly decreased number of colonies in the lung of mice given C26-6F or C26-8B tumors i.v. than in mice given mock C26 tumors i.v. Moreover, in mice given C26-6F cells i.v., a smaller number of tumor colonies in the lung was observed, as compared to the case with C26-6B cells. While the growth rate of C26-8B tumors in mice treated with anti-IL-5 mAb was more rapid than that seen in control mAb-treated mice, growth of C26-6F tumors in anti-IL-5-mAb-treated mice was slightly more rapid compared to findings in control mAb-treated mice. The isotypematched mAb did not alter the in vitro growth of mock-C26 cells or of the IL-5-gene-modified C26 cells. Growth of IL-5-secreting C26 tumors transplanted in nude mice was also inhibited. These results suggest that tumor-producing IL-5 inhibits growth of colon tumors mediated through T-cell-independent protective mechanisms of the host.     

敲低肿瘤细胞来源的颗粒体蛋白前体抑制C57BL/6J小鼠黑色素移植瘤的生长

颗粒体蛋白前体 (progranulin, PGRN)在多种肿瘤中过表达。但PGRN在黑色素瘤发生发展中的作用尚无报道。为探究PGRN在黑色素肿瘤中的作用,本研究采用CRISPR-Cas9基因编辑技术建立了稳定敲低PGRN的小鼠黑色素瘤B16细胞株B16-PGRNlow。MTS法和BrdU掺入结合流式细胞（计量）术分析证明,敲低PGRN不影响B16细胞的细胞周期和增殖。将B16-ctrl（对照）和B16-PGRNlow细胞分别皮下接种野生型（WT）和PGRN敲除（KO）的C57BL/6J小鼠,比较观察黑色素移植瘤体积大小。移植瘤形成20 d后,与B16-ctrl细胞接种的移植瘤比较,无论在WT还是在KO荷瘤小鼠,B16-PGRNlow形成的移植瘤体积明显减小（WT鼠：P<0.05;KO鼠：P<0.01）。然而,比较B16-PGRNlow或B16-ctrl在WT鼠与KO鼠形成的移植瘤体积大小,并无显著差异,提示B16肿瘤细胞PGRN而非宿主PGRN影响移植瘤的生长。流式细胞术分析显示,在荷B16-PGRNlow移植瘤的WT型小鼠脾和淋巴结中,CD4+、CD8+T细胞数（百分比）比荷B16-ctrl移植瘤的WT鼠脾和淋巴结的CD4+、CD8+T细胞数明显增多（P<0.05,P<0.01）,而在KO鼠却未见明显差异。上述结果证明,敲低肿瘤细胞PGRN可抑制黑色素移植瘤的生长。上述结果还提示,抑制PGRN在黑色瘤的表达可引起脾和淋巴结CD4+和CD8+T细胞增加,提高宿主的细胞免疫能力。其机制尚待进一步研究。本文的发现为PGRN作为黑色素瘤治疗的潜在靶点提供了新证据。     

Characterization and growth factor requirements of SJL lymphomas. I. Development of a B cell growth factor-dependent in vitro cell line, cRCS-X

Reticulum cell sarcomas (RCS) of SJL mice are completely dependent on host cells for their growth and therefore fail to grow in vitro. RCS cells induce marked proliferation in SJL Ly-1+2- T cells accompanied by lymphokine production. In an attempt to fully understand the host-tumor cell interaction, an RCS cell line, cRCS-X, was established in vitro from a transplantable tumor by the addition, every 3 wk, of gamma-irradiated syngeneic lymph node (LN) cells to the culture. cRCS-X maintains all of the characteristics of the parent tumor, RCS-X, including cell surface phenotype (Ks and I-As positive, Ds negative and B cell marker 14.8 positive), ability to stimulate host T cells, and ability to grow in nonirradiated but not in gamma-irradiated SJL mice. The growth factor requirements of cRCS-X were examined. It was found that human BCGF can replace gamma-irradiated LN cells in the maintenance of long term in vitro growth of cRCS-X. cRCS-X cells respond to human B cell growth factor (BCGF) or to recombinant murine interleukin (IL)-5 in a short term proliferation assay [( 3H]thymidine incorporation) in a dose-dependent manner in the presence and absence of fetal calf serum. BCGF also promotes colony formation in soft agar by cRCS-X cells. Although both IL-1 and interferon-gamma can synergize with BCGF in the induction of cRCS-X proliferation, these lymphokines, as well as IL-2, IL-3, granulocyte-macrophage colony-stimulating factor, and IL-4 have no effect on cRCS-X growth when added alone. In addition, it was shown that SJL LN cells produce both IL-4 and BCGF II activities as assayed on murine B cells, after stimulation with gamma-irradiated cRCS-X cells. In light of these results it is postulated that IL-5, [corrected] produced by syngeneic T cells [corrected] after stimulation with RCS, is essential for RCS growth, both in vitro and in vivo.     

In vivo natural antitumor resistance against murine EL-4 lymphoma cells in lethally irradiated syngeneic C57Bl/6 mice

Natural resistance has been detected in lethally irradiated C57Bl/6 (B6) mice inoculated intravenously with the ascites form of a syngeneic B6 leukemia. EL-4 cells were injected into lethally irradiated (800 R) B6 mice and tumor cell proliferation was evaluated by 125IUdR uptake in different organs 4 days after the challenge. Differential growth of lymphoma cells was observed when young mice were injected as compared with older mice and when mice were treated with agents known to interfere with natural resistance (e.g., poly(I:C), FLV-P, carrageenan, cyclophosphamide, high doses of irradiated cells). Similar results were obtained by measuring rapid clearance of 125IUdR-labeled EL-4 cells from lungs of intact B6 mice. In vivo cold competition studies, employing EL-4 and several other tumor lines of the same or different haplotype, showed that only EL-4 and RBL-5 cells were capable of inhibiting syngeneic resistance against EL-4 tumor. On the contrary, YAC-1 lymphoma cells, the most susceptible target to natural killer-mediated cytotoxicity in vitro, did not compete. These results suggest that EL-4 cells express membrane determinants not detectable on normal H-2b parental bone marrow cells and are susceptible to natural resistance against hemopoietic tumor cells in lethally irradiated syngeneic B6 mice.     

IL-22-mediated tumor growth reduction correlates with inhibition of ERK1/2 and AKT phosphorylation and induction of cell cycle arrest in the G2-M phase

IL-22 is a recently discovered cytokine of the IL-10 family that binds to a class II cytokine receptor composed of IL-22R1 and IL-10R2(c) and influences a variety of immune reactions. As IL-22 has also been shown to modulate cell cycle and proliferation mediators such as ERK1/2 and JNK, we studied the role of IL-22 in proliferation, apoptosis, and cell cycle regulation in EMT6 murine breast cancer cells in vitro and in vivo. In this study, we report that murine breast cancer cells express functional IL-22R as indicated by RT-PCR studies, immunoblotting, and STAT3 activation assays. Importantly, IL-22 exposure of EMT6 cells resulted in decreased levels of phosphorylated ERK1/2 and AKT protein kinases, indicating an inhibitory effect of IL-22 on signaling pathways promoting cell proliferation. Furthermore, IL-22 induced a cell cycle arrest of EMT6 cells in the G(2)-M phase. IL-22 reduced EMT6 cell numbers and the proliferation rate by approximately 50% as measured by [(3)H]thymidine incorporation. IL-22 treatment of EMT6 tumor-bearing mice lead to a decreased tumor size and a reduced tumor cell proliferation in vivo, as determined by 3'-deoxy-3'-fluorothymidine-positron emission tomography scans. Interestingly, IL-22 did not induce apoptosis, as determined in annexin V binding assay and caspase-3 activation assay and had no effect on angiogenesis in vivo. In conclusion, our results indicate that IL-22 reduced tumor growth by inhibiting signaling pathways such as ERK1/2 and AKT phosphorylation that promote tumor cell proliferation in EMT6 cells. Therefore, IL-22 may play a role in the control of tumor growth and tumor progression.     

Adoptively transferred antigen-specific T cells can be grown and maintained in large numbers in vivo for extended periods of time by intermittent restimulation with specific antigen plus IL-2

The aim of the current study was to determine whether cultured tumor Ag-specific T cells could be induced to grow and maintained functional in large numbers in vivo by intermittent restimulation in vivo with specific Ag plus IL-2. T cells derived from spleens of B6 mice (Thy-1.2) immune to FBL-3, a Friend virus-induced leukemia, were activated by in vitro stimulation with irradiated FBL-3 and expanded by culture for 14 days with low concentrations of IL-2. The resultant FBL-3-specific T cell lines were adoptively transferred into cyclophosphamide pretreated congenic hosts (B6/Thy-1.1), and restimulated every 14 days by an injection of irradiated FBL-3 plus a 7-day course of IL-2. Donor T cells residing in the host were identified and quantified by use of antibody to the Thy-1.2 allele. The results confirmed that stimulation with FBL-3 on the day of transfer (day 0) plus IL-2 on days 0 to 6 induced rapid growth of donor T cells to approximately an 11-fold increase in total donor T cell number recoverable from host ascites and spleen by day 7. However, prolonging the course of IL-2 administration to 35 days did not maintain the number or the specific cytolytic function of donor T cells. By contrast, intermittent restimulation with specific Ag plus IL-2 induced intermittent regrowth of donor T cells in vivo, maintained the number of donor T cells in vivo at greater than the number input for longer than 1 mo, and allowed detection of substantially augmented donor T cell-mediated specific antitumor function over that period of time.     

In vivo delivery of interleukin-6 using vaccinia virus: effects on T lymphocytes in nude mice

We have constructed a recombinant vaccinia virus (VV) expressing the human interleukin-6 (IL-6) gene, VV(IL-6). After injection of VV(IL-6) i.v. into Balb/c mice, circulating IL-6 was detected during 3 days with the peak activity on day 4, indicating that VV injection is an effective method to deliver lymphokines in vivo. We have further examined the effects of IL-6 in vivo in immunodeficient mice. Nude mice were injected i.v. with VV(IL-6). Ten days after the injection, mice were sacrificed and spleen cells were obtained. Spleen cells from VV(IL-6) injected mice proliferated remarkably in response to IL-2, while spleen cells from mice injected with unrelated VV manifested no particular proliferation in response to lymphokines. When spleen cells were further cultured in vitro for 5 days in the presence of Concanavalin-A stimulated rat spleen cell supernatant (Con-A factor), CD4 or CD8 positive cells were detected in the VV (IL-6) injected group, while few positive cells were detected in the control groups. These results suggest that IL-6 stimulates nude mice spleen cells in vivo, to a stage where they are able to proliferate in response to IL-2, or to differentiate into CD4 or CD8 positive cells in presence of rat Con-A factor.     

Identification of interleukin-6 as an autocrine growth factor for Epstein-Barr virus-immortalized B cells.

Autocrine growth factors are believed to be important for maintenance of an immortalized state by Epstein-Barr virus (EBV), because cell-free supernatants of EBV-immortalized cell lines promote the proliferation of autologous cells and permit their growth at low cell density. In this study, we provide evidence for the existence of two autocrine growth factor activities produced by EBV-immortalized lines distinguished by size and biological activities. Much of the autocrine growth factor activity in lymphoblastoid cell line supernatants resided in a low-molecular-weight (less than 5,000) fraction. However, up to 20 to 30% of the autocrine growth factor activity resided in the high-molecular-weight (greater than 5,000) fraction. While the nature of the low-molecular-weight growth factor activity remains undefined, the high-molecular-weight growth factor activity was identified as interleukin-6 (IL-6). Culture supernatants from six EBV-induced lymphoblastoid cell lines tested contained IL-6 activity, because they promoted proliferation in the IL-6-dependent hybridoma cell line B9. In addition, a rabbit antibody to human IL-6 neutralized the capacity of the high-molecular-weight (greater than 5,000) fraction of a lymphoblastoid cell line supernatant to promote growth both in autologous EBV-immortalized cells and in B9 cells. Similarly, this high-molecular-weight autocrine growth factor activity was neutralized by a monoclonal antibody to human IL-6. Furthermore, characteristic bands, attributable to IL-6, were visualized in supernatants of each of four EBV-induced lymphoblastoid cell lines after immunoprecipitation with a rabbit antiserum to human IL-6. Thus, in addition to its previously reported properties, IL-6 is an autocrine growth factor for EBV-immortalized B cells cultured under serum-free conditions.     

Tim-3+ T-bet+ tumor-specific Th1 cells colocalize with and inhibit development and growth of murine neoplasms

Although T cells infiltrate many types of murine and human neoplasms, in many instances tumor-specific cytotoxicity is not observed. Strategies to stimulate CTL-mediated antitumor immunity have included in vitro stimulation and/or genetic engineering of T cells, followed by adoptive transfer into tumor-bearing hosts. In this model of B cell lymphoma in SJL/J mice, we used Tim-3(+) T-bet(+) Th1 cells to facilitate the development of tumor-specific CTL. Tumor-specific Th1 cell lines were polarized with IL-12 during in vitro stimulation and long term maintenance. As few as 5 million Tim-3(+) T-bet(+) Th1 cells enabled recipients to resist growth of malignant transplantable cells. In addition, similar numbers of Th1 cells injected into 2- to 3-mo-old mice inhibited development of the spontaneous primary lymphomas, which normally arise in 90% of aging mice. CFSE(+) Th1 cells colocalized with injected tumor cells in vivo and formed conjugates with the tumor cells within follicles, whereas in nontumor-challenged recipients the CFSE(+) Th1 cells localized only within the T cell zones of the spleen. These results provide evidence that adoptive immunotherapy with Tim-3(+) T-bet(+) tumor-specific Th1 cells can be used to induce host cytotoxic responses that inhibit the development and growth of neoplastic cells.     

Long-term suppression of tumor growth by TNF requires a Stat1- and IFN regulatory factor 1-dependent IFN-gamma pathway but not IL-12 or IL-18

Tumor cells engineered to secrete TNF were used as a model to examine how persistently high local concentrations of TNF suppress tumor growth. TNF secretion had no effect on tumor cell proliferation in vitro but caused a very impressive growth arrest in vivo that was dependent on both bone marrow- and non-bone marrow-derived host cells expressing TNFR. Suppression also required an endogenous IFN-gamma pathway consisting minimally of IFN-gamma, IFN-gamma receptor, Stat1, and IFN regulatory factor 1 since mice with targeted disruption of any of the four genes failed to arrest tumor growth. The ability of these mice to suppress tumor growth was restored after they were reconstituted with bone marrow cells from Wt mice. Interestingly, mice lacking the major IFN-gamma-inducing cytokines IL-12 and IL-18 or T cells, B cells, and the majority of NK cells that are potential sources of IFN-gamma nevertheless inhibited tumor development. Moreover, multiple lines of evidence indicated that local release of IFN-gamma was not required to inhibit tumor formation. These results strongly suggest a novel function for the endogenous IFN-gamma pathway that without measurable IFN-gamma production or activity affects the ability of TNF to suppress tumor development.     

The role of adoptively transferred CD8 T cells and host cells in the control of the growth of the EG7 thymoma: factors that determine the relative effectiveness and homing properties of Tc1 and Tc2 effectors

We had previously examined the factors that regulate the response of OVA-specific TCR-transgenic CD8 T cells to the B16 OVA melanoma, growing as lung metastases. We examine here whether the same parameters operate for EG7, growing intradermally. Tc1 or Tc2 CD8 effector cells from OT-1 mice were injected either mixed with the tumor or i.v. at day 0 or 7. Tc2 were one-fifth to one-tenth as effective as Tc1 when injected with the tumor, in controlling tumor growth, but were only 1/20 to 1/100 injected i.v. Tc1 injected i.v. entered the draining lymph nodes faster than Tc2 and caused a faster accumulation of host cells. Both caused an abrupt termination of host cell entry into lymph nodes and spleen after tumor elimination, but this occurred earlier for Tc1 than for Tc2. Host responses were ineffective in the absence of adoptive transfer but were essential after transfer. Perforin expression in the donor cells plays no role in adoptively transferred Tc1 or Tc2 control of the tumor, and neither IL-4 nor IL5 is needed for Tc1 or Tc2 function. Tc1 cells from mice lacking IFN-gamma, however, control tumor growth less well, whereas Tc2 effectors lacking IFN-gamma are unaffected. Tc1 from IFN-gamma-deficient mice attract fewer host cells to the draining lymph node, whereas Tc1 cells from perforin-deficient donors are unimpaired. We conclude that host cell recruitment is a crucial element in adoptive immunotherapy. The differences between the EG7 and the previous B16 melanoma model are discussed.     

An essential role for macrophage migration inhibitory factor (MIF) in angiogenesis and the growth of a murine lymphoma.

BACKGROUND: Macrophage migration inhibitory factor (MIF) has been shown to counterregulate glucocorticoid action and to play an essential role in the activation of macrophages and T cells in vivo. MIF also may function as an autocrine growth factor in certain cell systems. We have explored the role of MIF in the growth of the 38C13 B cell lymphoma in C3H/HeN mice, a well-characterized syngeneic model for the study of solid tumor biology. MATERIALS AND METHODS: Tumor-bearing mice were treated with a neutralizing anti-MIF monoclonal antibody and the tumor response assessed grossly and histologically. Tumor capillaries were enumerated by immunohistochemistry and analyzed for MIF expression. The effect of MIF on endothelial cell proliferation was studied in vitro, utilizing both specific antibody and antisense oligonucleotide constructs. The role of MIF in angiogenesis also was examined in a standard Matrigel model of new blood vessel formation in vivo. RESULTS: The administration of anti-MIF monoclonal antibodies to mice was found to reduce significantly the growth and the vascularization of the 38C13 B cell lymphoma. By immunohistochemistry, MIF was expressed predominantly within the tumor-associated neovasculature. Cultured microvascular endothelial cells, but not 38C13 B cells, produced MIF protein and required its activity for proliferation in vitro. Anti-MIF monoclonal antibody also was found to markedly inhibit the neovascularization response elicited by Matrigel implantation. CONCLUSION: These data significantly expand the role of MIF in host responses, and suggest a new target for the development of anti-neoplastic agents that inhibit tumor neovascularization.     

Four cell-secreted cytokines act synergistically to maintain long term proliferation of human B cell lines in vitro.

Autocrine production of growth factors is thought to be an essential element in the development of hemopoietic tumors in vivo. Tumor-derived cell lines frequently show this capability in vitro. It is not understood how autonomous growth in vitro is maintained by lymphoid cell lines that are not of tumorigenic origin. We have previously established human B cell clones that proliferate in serum-free media with unlimited potential. However, the cells need a critical density for continuous growth. Culture supernatant conditioned by these cell lines sustained proliferation even in low density cultures. All B cell clones analyzed were found to secrete the cytokines IL-1 alpha, IL-6, TNF-alpha, and TNF-beta whereas no activity of IL-2, IL-4, low m. w.-B cell growth factor, CSF, or IFN-gamma was recorded. In low density cultures supplemented with rIL-1 alpha, +/- IL-6, +/- TNF-alpha, and +/- TNF-beta together, B cell proliferation is maintained to the same extent as with conditioned medium. Addition of anti-sense oligonucleotides directed to the mRNA of IL-1 alpha, IL-6, and TNF-alpha, respectively, resulted in growth arrest and cell death. This effect could be prevented by supplementation with these cytokines. Scatchard plot analyses and internalization studies revealed that the cells express on their surface high affinity receptors for IL-1 alpha, IL-6, and TNF, respectively, and internalize the cytokines from the supernatant. These results demonstrate that (i) autonomous growth of immortalized B cells is maintained by secretion and reinternalization of IL-1 alpha, IL-6, TNF-alpha, and TNF-beta, (ii) these cytokines act in a synergistic fashion, and (iii) autocrine growth stimulation of human B cells in vitro does not necessarily represent their tumorigenic potential in vivo.     

Accumulation of immunosuppressive CD11b+ myeloid cells correlates with the failure to prevent tumor growth in the anterior chamber of the eye

The purpose of these studies is to determine why an immunogenic tumor grows unchecked in the anterior chamber (a.c.) of the eye. The OVA-expressing EL4 tumor, E.G7-OVA, was injected into the a.c. or skin of immunocompetent and immunodeficient mice. Tumor growth and tumor-specific immune responses were monitored. Ocular tumor-infiltrating leukocytes were characterized phenotypically and functionally. Growth of E.G7-OVA was inhibited when limiting numbers of cells were injected in the skin but not in the a.c. of C57BL/6 mice, although both routes primed OVA-specific immune responses, which prevented the growth of a subsequent injection with E.G7-OVA in the skin or opposite eye. Tumor regression was OVA-specific because growth of the parental EL-4 tumor was not inhibited in primed mice. E.G7-OVA growth in the skin was not inhibited in immunodeficient Rag(-/-) or CD8 T cell-deficient mice, suggesting that CD8(+) CTLs mediate tumor elimination. CD8(+) T cell numbers were significantly increased in eyes of mice primed with E.G7-OVA, but few were detected in primary ocular tumors. Nevertheless, growth of E.G7-OVA was retarded in the a.c. of TCR-transgenic OT-I mice, and CD8(+) T cell numbers were increased within eyes, suggesting that tumor-specific CD8(+) CTLs migrated into and controlled primary ocular tumor growth. E.G7-OVA did not lose antigenicity or become immunosuppressive after 13 days of growth in the eye. However, CD11b(+) cells accumulated in primary ocular tumors and contained potent immunosuppressive activity when assayed in vitro. Thus, CD11b(+) cells that accumulate within the eye as tumors develop in the a.c. may contribute to immune evasion by primary ocular tumors by inhibiting CTLs within the eye.