Tumor-associated myeloid cells can be activated in vitro and in vivo to mediate antitumor effects

Tumor growth is often accompanied by the accumulation of myeloid cells in the tumors and lymphoid organs. These cells can suppress T cell immunity, thereby posing an obstacle to T cell-targeted cancer immunotherapy. In this study, we tested the possibility of activating tumor-associated myeloid cells to mediate antitumor effects. Using the peritoneal model of B16 melanoma, we show that peritoneal cells (PEC) in tumor-bearing mice (TBM) had reduced ability to secrete nitric oxide (NO) following in vitro stimulation with interferon gamma and lipopolysaccharide, as compared to PEC from control mice. This reduced function of PEC was accompanied by the influx of CD11b(+) Gr-1(+) myeloid cells to the peritoneal cavity. Nonadherent PEC were responsible for most of the NO production in TBM, whereas in na?ve mice NO was mainly secreted by adherent CD11b(+) F4/80(+) macrophages. Sorted CD11b(+) Gr-1(-) monocytic and CD11b(+) Gr-1(+) granulocytic PEC from TBM had a reduced ability to secrete NO following in vitro stimulation (compared to na?ve PEC), but effectively suppressed proliferation of tumor cells in vitro. In vivo, treatment of mice bearing established peritoneal B16 tumors with anti-CD40 and CpG resulted in activation of tumor-associated PEC, reduction in local tumor burden and prolongation of mouse survival. Inhibition of NO did not abrogate the antitumor effects of stimulated myeloid cells. Taken together, the results indicate that in tumor-bearing hosts, tumor-associated myeloid cells can be activated to mediate antitumor effects.     

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.     

Tumors suppress in situ proliferation of cytotoxic T cells by promoting differentiation of Gr-1(+) conventional dendritic cells through IL-6

Cancers are often accompanied by inflammation, which can promote tumor growth, invasion, and metastases. We show that the tumor microenvironment induces the development of a Gr-1(+) conventional dendritic cell (cDC) subpopulation that is functionally defective. Gr-1(+)cDCs differentiated from recruited immediate precursors of cDCs, a process supported by the inflammatory cytokine milieu in tumors. Inhibition of Gr-1(+)cDC differentiation enhanced intratumor expansion of cytotoxic CD8(+) T cells (CTLs), resulting in suppression of tumor growth. Diphtheria toxin treatment of CD11c-diphtheria toxin receptor chimeras revealed the importance of intratumor cDCs in stimulating CTL proliferation in situ. Our study demonstrates a key role of intratumor cDCs in determining antitumor CTL responses and suggests that they may be an appropriate target for tumor immunotherapy.     

Opposing roles for complement component c5a in tumor progression and the tumor microenvironment

Promoting complement (C) activation may enhance immunological mechanisms of anti-tumor Abs for tumor destruction. However, C activation components, such as C5a, trigger inflammation, which can promote tumor growth. We addressed the role of C5a on tumor growth by transfecting both human carcinoma and murine lymphoma with mouse C5a. In vitro growth kinetics of C5a, control vector, or parental cells revealed no significant differences. Tumor-bearing mice with C5a-transfected xenografted tumor cells had significantly less tumor burden as compared with control vector tumors. NK cells and macrophages infiltrated C5a-expressing tumors with significantly greater frequency, whereas vascular endothelial growth factor, arginase, and TNF-α production were significantly less. Tumor-bearing mice with high C5a-producing syngeneic lymphoma cells had significantly accelerated tumor progression with more Gr-1(+)CD11b(+) myeloid cells in the spleen and overall decreased CD4(+) and CD8(+) T cells in the tumor, tumor-draining lymph nodes, and the spleen. In contrast, tumor-bearing mice with low C5a-producing lymphoma cells had a significantly reduced tumor burden with increased IFN-γ-producing CD4(+) and CD8(+) T cells in the spleen and tumor-draining lymph nodes. These studies suggest concentration of local C5a within the tumor microenvironment is critical in determining its role in tumor progression.     

The importance of myeloid-derived suppressor cells in the regulation of autoimmune effector cells by a chronic contact eczema

Induction of a chronic eczema is a most efficient therapy for alopecia areata (AA). We had noted a reduction in regulatory T cells during AA induction and wondered whether regulatory T cells may become recruited or expanded during repeated skin sensitization or whether additional regulatory cells account for hair regrowth. AA could not be cured by the transfer of CD4(+)CD25(high) lymph node cells from mice repeatedly treated with a contact sensitizer. This obviously is a consequence of a dominance of freshly activated cells as compared with regulatory CD4(+)CD25(+) T cells. Instead, a population of Gr-1(+)CD11b(+) cells was significantly increased in skin and spleen of AA mice repeatedly treated with a contact sensitizer. Gr-1(+)CD11b(+) spleen cells mostly expressed CD31. Expression of several proinflammatory cytokines as well as of the IFN-gamma receptor and the TNF receptor I were increased. Particularly in the skin, Gr-1(+) cells expressed several chemokines and CCR8 at high levels. Gr-1(+)CD11b(+) cells most potently suppressed AA effector cell proliferation in vitro and promoted partial hair regrowth in vivo. When cocultured with CD4(+) or CD8(+) cells from AA mice, the Gr-1(+)CD11b(+) cells secreted high levels of NO. However, possibly due to high level Bcl-2 protein expression in AA T cells, apoptosis induction remained unaltered. Instead, zeta-chain expression was strongly down-regulated, which was accompanied by a decrease in ZAP70 and ERK1/2 phosphorylation. Thus, a chronic eczema supports the expansion and activation of myeloid suppressor cells that, via zeta-chain down-regulation, contribute to autoreactive T cell silencing in vitro and in vivo.     

Mechanism of immune dysfunction in cancer mediated by immature Gr-1+ myeloid cells

The mechanism of tumor-associated T cell dysfunction remains an unresolved problem of tumor immunology. Development of T cell defects in tumor-bearing hosts are often associated with increased production of immature myeloid cells. In tumor-bearing mice, these immature myeloid cells are represented by a population of Gr-1(+) cells. In this study we investigated an effect of these cells on T cell function. Gr-1(+) cells were isolated from MethA sarcoma or C3 tumor-bearing mice using cell sorting. These Gr-1(+) cells expressed myeloid cell marker CD11b and MHC class I molecules, but they lacked expression of MHC class II molecules. Tumor-induced Gr-1(+) cells did not affect T cell responses to Con A and to a peptide presented by MHC class II. In sharp contrast, Gr-1(+) cells completely blocked T cell response to a peptide presented by MHC class I in vitro and in vivo. Block of the specific MHC class I molecules on the surface of Gr-1(+) cells completely abrogated the observed effects of these cells. Thus, immature myeloid cells specifically inhibited CD8-mediated Ag-specific T cell response, but not CD4-mediated T cell response. Differentiation of Gr-1(+) cells in the presence of growth factors and all-trans retinoic acid completely eliminated inhibitory potential of these cells. This may suggest a new approach to cancer treatment.     

Human CD4+ effector memory T cells persisting in the microenvironment of lung cancer xenografts are activated by local delivery of IL-12 to proliferate, produce IFN-gamma, and eradicate tumor cells

The implantation of small pieces of human primary lung tumor biopsy tissue into SCID mice results in a viable s.c. xenograft in which the tissue architecture, including tumor-associated leukocytes, tumor cells, and stromal cells, is preserved in a functional state. By monitoring changes in tumor volume, gene expression patterns, cell depletion analysis, and the use of function-blocking Abs, we previously established in this xenograft model that exogenous IL-12 mobilizes human tumor-associated leukocytes to kill tumor cells in situ by indirect mechanisms that are dependent upon IFN-gamma. In this study immunohistochemistry and FACS characterize the early cellular events in the tumor microenvironment induced by IL-12. By 5 days post-IL-12 treatment, the constitutively present human CD45(+) leukocytes have expanded and infiltrated into tumor-rich areas of the xenograft. Two weeks post-treatment, there is expansion of the human leukocytes and complete effacement of the tumor compared with tumor progression and gradual loss of most human leukocytes in control-treated xenografts. Immunohistochemical analyses reveal that the responding human leukocytes are primarily activated or memory T cells, with smaller populations of B cells, macrophages, plasma cells, and plasmacytoid dendritic cells capable of producing IFN-alpha. The predominant cell population was also characterized by FACS and was shown to have a phenotype consistent with a CD4(+) effector memory T cell. We conclude that quiescent CD4(+) effector memory T cells are present within the tumor microenvironment of human lung tumors and can be reactivated by the local and sustained release of IL-12 to proliferate and secrete IFN-gamma, leading to tumor cell eradication.     

Nitric oxide-independent CTL suppression during tumor progression: association with arginase-producing (M2) myeloid cells

Most of the mice bearing a s.c. BW-Sp3 lymphoma tumor mount a CD8(+) T cell-mediated response resulting in tumor regression. Nonetheless, tumor progression occurs in some of the recipients and is associated with CTL inactivity. We demonstrated that T cell-activating APC were induced in regressors whereas T cell suppressive myeloid cells predominated in the spleen of progressors. Indeed, in vitro depletion of either the adherent or the CD11b(+) populations restored T cell cytotoxicity and proliferation in these mice. This CTL inhibition was cell-to-cell contact-dependent but not mediated by NO. However, the same progressor suppressive cells prevented the activity of in vitro-restimulated CTLs derived from regressors in a cell-to-cell contact and NO-dependent fashion. Thus, either the NO-dependent or -independent suppressive pathway prevailed, depending on the target CTL population. In addition, the suppressive population expressed a high arginase activity, suggesting an association of the suppressive phenotype with alternatively activated (M2) myeloid cells. However, the high arginase activity is not directly involved in the suppressive process. Our results provide new insights for myeloid cell-mediated CTL inhibition during cancer progression.     

The generation of both T killer and Th cell clones specific for the tumor-associated antigen HER2 using retrovirally transduced dendritic cells

Induction of antitumor immunity involves the presence of both CD8(+) CTLs and CD4(+) Th cells specific for tumor-associated Ags. Attempts to eradicate cancer by adoptive T cell transfer have been limited due to the difficulty of generating T cells with defined Ag specificity. The current study focuses on the generation of CTL and Th cells against the tumor-associated Ag HER2 using autologous dendritic cells (DC) derived from CD34(+) hematopoietic progenitor cells which have been retrovirally transduced with the human epidermal growth factor receptor 2 (HER2) gene. HER2-transduced DC elicited HER2-specific CD8(+) CTL that lyse HER2-overexpressing tumor cells in context of distinct HLA class I alleles. The induction of both HLA-A2 and -A3-restricted HER2-specific CTL was verified on a clonal level. In addition, retrovirally transduced DC induced CD4(+) Th1 cells recognizing HER2 in context with HLA class II. HLA-DR-restricted CD4(+) T cells were cloned that released IFN-gamma upon stimulation with DC pulsed with the recombinant protein of the extracellular domain of HER2. These data indicate that retrovirally transduced DC expressing the HER2 molecule present multiple peptide epitopes and subsequently elicit HER2-specific CTL and Th1 cells. The method of stimulating HER2-specific CD8(+) and CD4(+) T cells with retrovirally transduced DC was successfully implemented for generating HER2-specific CTL and Th1 clones from a patient with HER2-overexpressing breast cancer. The ability to generate and expand HER2-specific, HLA-restricted CTL and Th1 clones in vitro facilitates the development of immunotherapy regimens, in particular the adoptive transfer of both autologous HER2-specific T cell clones in patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.     

Immortalized myeloid suppressor cells trigger apoptosis in antigen-activated T lymphocytes

We described a generalized suppression of CTL anamnestic responses that occurred in mice bearing large tumor nodules or immunized with powerful recombinant viral immunogens. Immune suppression entirely depended on GM-CSF-driven accumulation of CD11b(+)/Gr-1(+) myeloid suppressor cells (MSC) in secondary lymphoid organs. To further investigate the nature and properties of MSC, we immortalized CD11b(+)/Gr-1(+) cells isolated from the spleens of immunosuppressed mice, using a retrovirus encoding the v-myc and v-raf oncogenes. Immortalized cells expressed monocyte/macrophage markers (CD11b, F4/80, CD86, CD11c), but they differed from previously characterized macrophage lines in their capacities to inhibit T lymphocyte activation. Two MSC lines, MSC-1 and MSC-2, were selected based upon their abilities to inhibit Ag-specific proliferative and functional CTL responses. MSC-1 line was constitutively inhibitory, while suppressive functions of MSC-2 line were stimulated by exposure to the cytokine IL-4. Both MSC lines triggered the apoptotic cascade in Ag-activated T lymphocytes by a mechanism requiring cell-cell contact. Some well-known membrane molecules involved in the activation of apoptotic pathways (e.g., TNF-related apoptosis-inducing ligand, Fas ligand, TNF-alpha) were ruled out as candidate effectors for the suppression mechanism. The immortalized myeloid lines represent a novel, useful tool to shed light on the molecules involved in the differentiation of myeloid-related suppressors as well as in the inhibitory pathway they use to control T lymphocyte activation.     

Inflammatory blood monocytes contribute to tumor development and represent a privileged target to improve host immunosurveillance

Progressing tumors in humans and mice are frequently infiltrated by a highly heterogeneous population of inflammatory myeloid cells that contribute to tumor growth. Among these cells, inflammatory Gr-1(+) monocytes display a high developmental plasticity in response to specific microenvironmental signals, leading to diverse immune functions. These observations raise the question of the immune mechanisms by which inflammatory monocytes may contribute to tumor development. In this study, we found that adoptive transfer of normal inflammatory Gr-1(+) monocytes in tumor-bearing mice promotes tumor growth. In this tumoral environment, these monocytes can differentiate into tolerogenic dendritic cells (DCs) that produce IL-10 and potently induce regulatory T cell responses in vivo. Moreover, diverting the differentiation of Gr-1(+) monocytes into tolerogenic DCs by forced expression of IL-10 soluble receptor and IL-3 in tumor cells improves host immunosurveillance by reducing the regulatory T cell frequency and by inducing immunogenic DCs in the tumor. As a consequence, tumor growth is strongly reduced. Our findings indicate that Gr-1(+) monocytes represent a valuable target for innovative immunotherapeutic strategies against cancer.     

ISCOMATRIX adjuvant combines immune activation with antigen delivery to dendritic cells in vivo leading to effective cross-priming of CD8+ T cells

Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-γ production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8α(+) DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8α(+) DCs in VDLNs, whereas Langerin(+) DCs and CD8α(-) DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8α(+) DCs in vivo for efficient induction of CTL responses.     

Adenosinergic regulation of the expansion and immunosuppressive activity of CD11b+Gr1+ cells

Extracellular adenosine and purine nucleotides are elevated in many pathological situations associated with the expansion of CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSCs). Therefore, we tested whether adenosinergic pathways play a role in MDSC expansion and functions. We found that A(2B) adenosine receptors on hematopoietic cells play an important role in accumulation of intratumoral CD11b(+)Gr1(high) cells in a mouse Lewis lung carcinoma model in vivo and demonstrated that these receptors promote preferential expansion of the granulocytic CD11b(+)Gr1(high) subset of MDSCs in vitro. Flow cytometry analysis of MDSCs generated from mouse hematopoietic progenitor cells revealed that the CD11b(+)Gr-1(high) subset had the highest levels of CD73 (ecto-5'-nucleotidase) expression (Δmean fluorescence intensity [MFI] of 118.5 ± 16.8), followed by CD11b(+)Gr-1(int) (ΔMFI of 57.9 ± 6.8) and CD11b(+)Gr-1(-/low) (ΔMFI of 12.4 ± 1.0) subsets. Even lower levels of CD73 expression were found on Lewis lung carcinoma tumor cells (ΔMFI of 3.2 ± 0.2). The high levels of CD73 expression in granulocytic CD11b(+)Gr-1(high) cells correlated with high levels of ecto-5'-nucleotidase enzymatic activity. We further demonstrated that the ability of granulocytic MDSCs to suppress CD3/CD28-induced T cell proliferation was significantly facilitated in the presence of the ecto-5'-nucleotidase substrate 5'-AMP. We propose that generation of adenosine by CD73 expressed at high levels on granulocytic MDSCs may promote their expansion and facilitate their immunosuppressive activity.     

A spontaneously arising pancreatic tumor does not promote the differentiation of naive CD8+ T lymphocytes into effector CTL

In this report, we address whether a growing tumor provides sufficient inflammatory signals to promote activation, clonal expansion, and acquisition of effector functions by naive tumor-specific CD8(+) T lymphocytes. CD8(+) T lymphocytes obtained from hemagglutinin (HA)-specific clone 4 TCR-transgenic mice were injected into recipient mice that spontaneously develop pancreatic tumors expressing HA as a tumor-associated Ag (RIP-Tag2-HA mice). When 3 x 10(6) clone 4 CD8(+) T cells were transferred into tumor-bearing mice, the cells became activated in the pancreatic lymph nodes where they proliferated and acquired effector functions such as cytolytic activity and IFN-gamma production. Surprisingly, reducing the number of adoptively transferred CD8(+) T cells led to a parallel reduction in the proportion of the activated cells that exhibited effector functions, suggesting that CTL differentiation was induced by the large numbers of activated CD8(+) T cells and not the tumor environment. Provision of tumor-specific CD4(+) helper cells provided the signals required to promote both the development of CTL effector functions and increased clonal expansion, resulting in tumor eradication. Considering that only small numbers of tumor-specific CD8(+) T cells would be present in a conventional T cell repertoire, these data suggest that tumor growth alone may not provide the inflammatory signals necessary to support the development of CD8(+) T cell effector functions.     

Tumor-conditioned Gr-1CD11b myeloid cells induce angiogenesis through the synergistic action of CCL2 and CXCL16 in vitro

Gr-1⁺CD11b⁺ cells can suppress innate and adaptive immunity, and the functional immunosuppressive characteristics of these cells can be modulated by the tumor microenvironment. Since Gr-1⁺CD11⁺ cells are also involved in tumor-associated angiogenesis, we hypothesized that the angiogenic nature of Gr-1⁺CD11b⁺ cells could be regulated by the tumor milieu. To address this hypothesis, we imitated a tumor microenvironment by exposing Gr-1⁺CD11b⁺ cells isolated from spleen of 4T1 mammary carcinoma-bearing mice to tumor-conditioned medium. Supernatants from tumor-conditioned Gr-1⁺CD11b⁺ cells significantly induced capillary-like tube formation and migration of human umbilical vein endothelial cells (HUVECs) compared to naive Gr-1⁺CD11b⁺ cells. Incubation of Gr-1⁺CD11b⁺ cells with tumor-conditioned medium induced production of pro-angiogenic chemokines CCL2 and CXCL16. Pretreatment with an anti-CCL2 antibody, but not an anti-CXCL16 antibody, suppressed the angiogenic effects of tumor-conditioned Gr-1⁺CD11b⁺ cells on HUVECs. Simultaneous neutralization of CCL2 and CXCL16 significantly inhibited tube formation and migration of HUVECs compared to the sole neutralization against CCL2. Supernatants from tumor-conditioned Gr-1⁺CD11b⁺ cells induced phosphorylation of ERK1/2 in HUVECs, and inhibition of the ERK pathway blocked angiogenic effects. ERK pathway activity was partially abrogated by neutralization of CCL2 and more suppressed by simultaneous neutralization of CCL2 and CXCL16. These results collectively indicate that CCL2 and CXCL16 chemokines produced by tumor-conditioned Gr-1⁺CD11b⁺ myeloid cells synergistically induce angiogenesis in vitro by stimulating the ERK1/2 signaling pathway. Thus, regulation of Gr-1⁺CD11b⁺ cells in the tumor microenvironment may contribute to angiogenesis through the secretion of pro-angiogenic chemokines.     

Tumor-infiltrating programmed death receptor-1+ dendritic cells mediate immune suppression in ovarian cancer

Within the ovarian cancer microenvironment, there are several mechanisms that suppress the actions of antitumor immune effectors. Delineating the complex immune microenvironment is an important goal toward developing effective immune-based therapies. A dominant pathway of immune suppression in ovarian cancer involves tumor-associated and dendritic cell (DC)-associated B7-H1. The interaction of B7-H1 with PD-1 on tumor-infiltrating T cells is a widely cited theory of immune suppression involving B7-H1 in ovarian cancer. Recent studies suggest that the B7-H1 ligand, programmed death receptor-1 (PD-1), is also expressed on myeloid cells, complicating interpretations of how B7-H1 regulates DC function in the tumor. In this study, we found that ovarian cancer-infiltrating DCs progressively expressed increased levels of PD-1 over time in addition to B7-H1. These dual-positive PD-1(+) B7-H1(+) DCs have a classical DC phenotype (i.e., CD11c(+)CD11b(+)CD8(-)), but are immature, suppressive, and respond poorly to danger signals. Accumulation of PD-1(+)B7-H1(+) DCs in the tumor was associated with suppression of T cell activity and decreased infiltrating T cells in advancing tumors. T cell suppressor function of these DCs appeared to be mediated by T cell-associated PD-1. In contrast, ligation of PD-1 expressed on the tumor-associated DCs suppressed NF-κB activation, release of immune regulatory cytokines, and upregulation of costimulatory molecules. PD-1 blockade in mice bearing ovarian cancer substantially reduced tumor burden and increased effector Ag-specific T cell responses. Our results reveal a novel role of tumor infiltrating PD-1(+)B7-H1(+) DCs in mediating immune suppression in ovarian cancer.     

Reduction of myeloid-derived suppressor cells and induction of M1 macrophages facilitate the rejection of established metastatic disease

More than 60% of STAT6(-/-) mice immunologically reject spontaneous metastatic mammary carcinoma and survive indefinitely if their primary tumors are removed, whereas 95% of STAT6-competent BALB/c mice succumb to metastatic disease. BALB/c and STAT6-deficient mice with primary tumors have elevated levels of Gr1(+)CD11b(+) myeloid suppressor cells (MSCs), which inhibit T cell activation. After removal of primary tumor, MSC levels revert to baseline in STAT6-deficient mice, but remain elevated in BALB/c mice. The decrease is IFN-gamma dependent, as is the reduction in metastatic disease. Neither BALB/c nor STAT6-deficient MSCs produce inducible NO synthase; however, both produce arginase and reactive oxygen species. STAT6-deficient mice produce M1 macrophages, which contain high levels of NO and are tumoricidal, whereas BALB/c mice produce M2 macrophages, which make arginase and are not tumoricidal. Immunity in STAT6-deficient mice requires the activation of NO-producing M1 macrophages that are tumoricidal, the reduction in MSC levels to baseline after surgical removal of primary tumor, and the activation of tumor-specific T cells. These mechanisms occur in STAT6(-/-) mice because STAT6 deficiency prevents signaling through the type 2 IL-4Ralpha, thereby blocking the production of arginase and promoting the synthesis of NO.     

IL-17-dependent, IFN-gamma-independent tumor rejection is mediated by cytotoxic T lymphocytes and occurs at extraocular sites, but is excluded from the eye

Although intraocular tumors reside in an immune-privileged site where immune responses are suppressed, some tumors are rejected. An example of this is the rejection of intraocular adenovirus-induced (adenovirus type 5 early region 1 [Ad5E1]) tumors in C57BL/6 mice. We previously identified an Ad5E1 tumor clone in which the rejection is IFN-γ dependent and culminates in the destruction of both the tumor and the eye. Although Ad5E1 tumors are not rejected when transplanted into the eyes of IFN-γ KO mice, they are rejected after s.c. transplantation. Thus, outside of the eye Ad5E1 tumors elicit a form of tumor immunity that is IFN-γ independent. In this article, we demonstrate that IFN-γ-independent s.c. rejection requires both CD4(+) and CD8(+) T cells. Furthermore, s.c. tumor rejection requires IL-17, which is produced by IFN-γ-deficient CD4(+) T cells in response to tumor Ags (TAs). Splenocytes from CD4-depleted IFN-γ KO mice produce significantly less IL-17 compared with splenocytes from isotype-treated IFN-γ KO animals in response to TAs. Furthermore, depletion of IL-17 decreases CTL activity against Ad5E1 tumor cells. In this model we propose that, in the absence of IFN-γ, CD4(+) T cells produce IL-17 in response to TAs, which increases CTL activity that mediates tumor rejection; however, this does not occur in the eye. IL-6 production within the eye is severely reduced, which is consistent with the failure to induce Th17 cells within the intraocular tumors. In contrast, the s.c. environment is replete with IL-6 and supports the induction of Th17 cells. Therefore, IFN-γ-independent tumor rejection is excluded from the eye and may represent a newly recognized form of ocular immune privilege.     

Identification of tumor-infiltrating macrophages as the killers of tumor cells after immunization in a rat model system.

Immunization can prevent tumor growth, but the effector cells directly responsible for tumor cell killing in immunized hosts remain undetermined. The present study compares tumor grafts that progress in naive syngeneic rats with the same grafts that completely regress in hosts preimmunized with an immunogenic cell variant. The progressive tumors contain only a few macrophages that remain at the periphery of the tumor without direct contact with the cancer cells. These macrophages do not kill tumor cells in vitro. In contrast, tumors grafted in immunized hosts and examined at the beginning of tumor regression show a dramatic infiltration with mature macrophages, many of them in direct contact with the cancer cells. These macrophages are strongly cytotoxic for the tumor cells in vitro. In contrast to macrophages, tumor-associated lymphocytes are not directly cytotoxic to the tumor cells, even when obtained from tumor-immune rats. However, CD4(+) and CD8(+) T cells prepared from the regressing tumors induce tumoricidal activity in splenic macrophages from normal or tumor-bearing rats and in macrophages that infiltrate progressive tumors. These results strongly suggest that the main tumoricidal effector cells in preimmunized rats are macrophages that have been activated by adjacent tumor-immune lymphocytes.     

Long-term engraftment and expansion of tumor-derived memory T cells following the implantation of non-disrupted pieces of human lung tumor into NOD-scid IL2Rgamma(null) mice

Non-disrupted pieces of primary human lung tumor implanted into NOD-scid IL2Rgamma(null) mice consistently result in successful xenografts in which tissue architecture, including tumor-associated leukocytes, stromal fibroblasts, and tumor cells are preserved for prolonged periods with limited host-vs-graft interference. Human CD45(+) tumor-associated leukocytes within the xenograft are predominantly CD3(+) T cells with fewer CD138(+) plasma cells. The effector memory T cells that had been shown to be quiescent in human lung tumor microenvironments can be activated in situ as determined by the production of human IFN-gamma in response to exogenous IL-12. Plasma cells remain functional as evidenced by production of human Ig. Significant levels of human IFN-gamma and Ig were detected in sera from xenograft-bearing mice for up to 9 wk postengraftment. Tumor-associated T cells were found to migrate from the microenvironment of the xenograft to the lung, liver, and primarily the spleen. At 8 wk postengraftment, a significant portion of cells isolated from the mouse spleens were found to be human CD45(+) cells. The majority of CD45(+) cells were CD3(+) and expressed a phenotype consistent with an effector memory T cell, consisting of CD4(+) or CD8(+) T cells that were CD45RO(+), CD44(+), CD62L(-), and CD25(-). Following adoptive transfer into non-tumor bearing NOD-scid IL2Rgamma(null) mice, these human T cells were found to expand in the spleen, produce IFN-gamma, and maintain an effector memory phenotype. We conclude that the NOD-scid IL2Rgamma(null) tumor xenograft model provides an opportunity to study tumor and tumor-stromal cell interactions in situ for prolonged periods.