Migration of cytotoxic T lymphocytes toward melanoma cells in three-dimensional organotypic culture is dependent on CCL2 and CCR4

Studies in experimental animal models have demonstrated that chemokines produced by tumor cells attract chemokine receptor-positive T lymphocytes into the tumor area. However, in cancer patients, the role of chemokines in T lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the migration of a melanoma patient's CTL toward autologous tumor cells has been studied in a novel three-dimensional organotypic melanoma culture. In this model, CTL migrated toward tumor cells, resulting in tumor cell apoptosis. CTL migration was mediated by the CC chemokine receptor (CCR)4 expressed by the CTL and the CC chemokine ligand (CCL)2 secreted by the tumor cells, as evidenced by blockage of CTL migration by CCL2 or antibodies to CCL2 or CCR4. These results were confirmed in a Transwell migration assay in which the CTL actively migrated toward isolated CCL2 and migration was inhibited by anti-CCR4 antibody. These studies, together with previous studies in mice indicating regression of CCL2-transduced tumor cells, suggest that CCL2 may be useful as an immunotherapeutic agent for cancer patients.     

Secretion of CXC chemokine IP-10 by peripheral blood mononuclear cells from healthy donors and breast cancer patients stimulated with HER-2 peptides.

CXC chemokines play an important role in recruitment of T cells to the site of activation and regulation of angiogenesis. CXC chemokines are secreted by T cells stimulated with cytokines or by established cytotoxic T lymphocyte (CTL) lines at recognition of conventional antigen (Ag), but the activation requirements and the relationship of interferon-gamma (IFN-gamma) inducible protein (IP-10) secretion with IFN-gamma induction in lymphocytes are still unclear. We studied the induction of IP-10 from nonadherent peripheral blood mononuclear cells (PBMC) by IFN-gamma, interleukin-12 (IL-12), and the HER-2 peptide E75, which forms a CTL-defined antigen. We found that IFN-gamma alone was a weak inducer of IP-10 in these cells, whereas IL-12 was a significantly stronger inducer of IP-10. In the presence of IL-12, the tumor peptide E75 (HER-2, 369-377) was a stronger inducer of IP-10 than was IL-12 alone. E75 and its variants mutated at position 5 could also induce IP-10 in the absence of exogenous IL-12 or IFN-gamma. IP-10 induction by E75 required HLA-A2 presentation and B7-CD28 interactions and was partially inhibited by blocking of CD40-CD40L interactions. These results indicate that presentation of tumor peptides to peripheral T cells can induce a fast chemokine response, which in its early phase may be higher than the IFN-gamma response. This shows that the IP-10 response was independent of any early-phase IFN-gamma response in peripheral T cells. This may be important for understanding the regulation of the balance between chemoattractant chemokines (CC) and CXC chemokines by tumor Ag and may have implications for understanding the mechanisms of polarization of T cells and conditioning of antigen-presenting cells (APC) by tumor antigens.     

表达前列腺特异性膜抗原的DNA疫苗对肿瘤细胞的抑制作用

目的构建表达前列腺特异性膜抗原（PSMA）的DNA疫苗，观察其在体外对肿瘤细胞的免疫攻击和在体内对肿瘤细胞攻击的免疫保护作用。方法通过稳定转染构建表达PSMA的小鼠黑色素瘤细胞系B16-PSMA，将DNA疫苗pCDNA3．1-PSMA通过肌肉注射导入C57BL／6小鼠体内，分离小鼠脾细胞，检测细胞毒性T淋巴细胞（cytotoxic T lymphocytes，CTL）反应。以B16-PSMA细胞攻击免骺小鼠，观察免疫动物的无瘤生存期和肿瘤体积增长情况，评价DNA疫苗的抗肿瘤作用。结果DNA疫苗可诱导小鼠脾淋巴细胞CTL活性，经过免疫后的小鼠成瘤率降低，无瘤生存期延长，肿瘤生长缓慢，肿瘤组织内有较多淋巴细胞浸润，表明产生较强的抗肿瘤反应。结论表达PSMA的DNA疫苗能够诱导小鼠产生特异性免疫反应，对表达PSMA的肿瘤细胞的攻击产生免疫保护作用，为前列腺癌的预防和免疫治疗提供了新的思路。     

Regulation of inflammatory factors by double-stranded RNA receptors in breast cancer cells

Malignant cells are not the only components of a tumor mass since other cells (e.g., fibroblasts, infiltrating leukocytes and endothelial cells) are also part of it. In combination with the extracellular matrix, all these cells constitute the tumor microenvironment. In the last decade the role of the tumor microenvironment in cancer progression has gained increased attention and prompted efforts directed to abrogate its deleterious effects on anti-cancer therapies. The immune system can detect and attack tumor cells, and tumor-infiltrating lymphocytes (particularly CD8?T cells) have been associated with improved survival or better response to therapies in colorectal, melanoma, breast, prostate and ovarian cancer patients among others. Contrariwise, tumor-associated myeloid cells (myeloid-derived suppressor cells [MDSCs], dendritic cells [DCs], macrophages) or lymphoid cells such as regulatory T cells can stimulate tumor growth via inhibition of immune responses against the tumor or by participating in tumor neoangiogenesis.Herewith we analyzed the chemokine profile of mouse breast tumors regarding their capacity to generate factors capable of attracting and sequestering DCs to their midst. Chemoattractants from tumors were investigated by molecular biology and immunological techniques and tumor infiltrating DCs were investigated for matched chemokine receptors. In addition, we investigated the inflammatory response of breast cancer cells, a major component of the tumor microenvironment, to double-stranded RNA stimulation. By using molecular biology techniques such as qualitative and quantitative PCR, PCR arrays, and immunological techniques (ELISA, cytokine immunoarrays) we examined the effects of dsRNA treatment on the cytokine secretion profiles of mouse and human breast cancer cells and non-transformed cells.We were able to determine that tumors generate chemokines that are able to interact with receptors present on the surface of tumor infiltrating DCs. We observed that PRR signaling is able to modify the production of chemokines by breast tumor cells and normal breast cells, thereby constituting a possible player in shaping the profile of the leukocyte population in the TME.     

Immunodominant deters the response to other tumor antigens thereby favoring escape: prevention by vaccination with tumor variants selected with cloned cytolytic T cells in vitro

Variant cancer cells which arise from the parent tumor during tumor progression can escape immunity but retain antigens. We have mixed highly immunogenic (A⁺B⁺) murine parental cancer cells with less immunogenic (A^-B⁺) variant cancer cells to construct a model of a cancer containing escape variants. When such mixtures of cancer cells were injected into normal mice, the variant cells grew out because immune responsiveness to the B antigen on the variant was hindered by dominance of the A antigen on the surrounding parental tumor cells. However, A^-B⁺ variant cells inoculated alone at a separate site induced B specific cytolytic T cells and were rejected. Moreover, mice immunized with A^-B⁺ cells rejected a challenge which contained a mixture of variant and parental cancer cells, while immunization with A⁺B⁺ cells was ineffective. Thus, variant tumor cells selected from parental tumor cells by cytolytic T cells in vitro can be used to induce protective immunity against variants expected to escape tumor immunity in vivo. The immunodominance of the A antigen may be related to its ability to induce a much more rapid CTL response than the B antigen, since we show in another model that the preexistence of a CTL response to one antigen prevented the subsequent induction of CTL to another antigen injected at the same site, even if both antigens were equally efficient at inducing CTL. These results indicate that immunodominance can affect strong as well as weak antigens. Vaccination with individual antigens at separate sites rather than with multiple antigens at one site may, therefore, be needed to prevent tumor escape and tumor recurrence or to counteract infectious diseases.     

SDF-1/CXCR4生物学轴与肿瘤关系的研究进展

趋化因子SDF-1,又称CXCL12,PBSF,与其特异性受体CXCR4广泛表达在多种组织和器官上,它们所构成的SDF-1/CXCR4 生物学轴在多种肿瘤的发生,发展以及转移中都发挥重要作用,其可能是通过MAPK,AKT通路发挥作用。对这一特殊生物学轴的研究可能为肿瘤防治找到新的突破口。     

<Emphasis Type="Italic">In vitro</Emphasis> migration of cytotoxic T lymphocyte derived from a colon carcinoma patient is dependent on CCL2 and CCR2

Background  

Infiltration of colorectal carcinomas (CRC) with T-cells has been associated with good prognosis. There are some indications that chemokines could be involved in T-cell infiltration of tumors. Selective modulation of chemokine activity at the tumor site could attract immune cells resulting in tumor growth inhibition. In mouse tumor model systems, gene therapy with chemokines or administration of antibody (Ab)-chemokine fusion proteins have provided potent immune mediated tumor rejection which was mediated by infiltrating T cells at the tumor site. To develop such immunotherapeutic strategies for cancer patients, one must identify chemokines and their receptors involved in T-cell migration toward tumor cells.     

The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells.

To avoid detection by CTL, HIV encodes mechanisms for removal of class I MHC proteins from the surface of infected cells. However, class I downregulation potentially exposes the virus-infected cell to attack by NK cells. Human lymphoid cells are protected from NK cell cytotoxicity primarily by HLA-C and HLA-E. We present evidence that HIV-1 selectively downregulates HLA-A and HLA-B but does not significantly affect HLA-C or HLA-E. We then identify the residues in HLA-C and HLA-E that protect them from HIV down-regulation. This selective downregulation allows HIV-infected cells to avoid NK cell-mediated lysis and may represent for HIV a balance between escape from CTL and maintenance of protection from NK cells. These results suggest that subpopulations of CTL and NK cells may be uniquely suited for combating HIV.     

The diverse role of chemokines in tumor progression: prospects for intervention (Review)

Chemokines, proteins chemotactic for leukocytes and non-leukocytes, have been intensively studied for their role in tumor growth and metastasis. Recent work has shown that particular chemokines may have multiple effects on tumors including promoting growth, angiogenesis, metastasis, and suppression of the immune response to cancer, while other chemokines inhibit tumor mediated angiogenesis and promote anti-tumor immune responses. Increasing biological evidence supports the hypothesis that tumor-generated chemokines provide more than simply angiogenic signals. Tumor-derived chemokines may potentially act as inhibitors of anti-tumor immune responses as well as autocrine growth factors for the tumor. The complexity and redundancy of tumor chemokine expression suggests that a single chemokine target for tumor therapy may not be appropriate. Indeed, multiple target therapy including blockade of tumor enhancing chemokines while delivering or inducing the secretion of anti-tumor chemokines is the approach that currently holds the most promise. The role of chemokines in tumor biology as well as various means of blocking chemokines in cancer models in order to develop successful therapeutic strategies will be discussed.     

Chemokines and their role in tumor growth and metastasis

Chemokines are a superfamily of pro-inflammatory polypeptide cytokines that selectively attract and activate different cell types. Many patho-physiological conditions require the participation of chemokines, including inflammation, infection, tissue injury, allergy, cardiovascular diseases, as well as malignant tumors. Chemokines activate cells through their binding to shared or unique cell surface receptors which belong to the seven-transmembrane, G-protein-coupled Rhodopsin superfamily. The role of chemokines in malignant tumors is complex: while some chemokines may enhance innate or specific host immunity against tumor implantation, others may favor tumor growth and metastasis by promoting tumor cell proliferation, migration or neovascularization in tumor tissue. In this review, the authors summarize some of the recent advances in chemokine research and emphasis is made on the effect of chemokines in tumor growth and metastasis.     

TH细胞在记忆性CTL介导的肿瘤抑制中的作用研究

目的 观察记忆性CTL的抑瘤作用是否需要抗原特异性TH细胞的辅助。方法 表达OVAT细胞表位SIINFEKL特异性TCR的T细胞转输RAG^-l-小鼠，经相应T细胞表位多肽刺激后产生记忆性CTL，将此记忆性CTL转输已产生特异性和非特异性TH细胞的C57BL／6小鼠体内并接种肿瘤细胞EG7。结果 经T细胞表位多肽免疫后，SIINFEKL特异性TCR T细胞成功在RAG^-l-小鼠体内增殖并分化为记忆性CTL；抗原特异性TH细胞可辅助产生较多效应性CTL但并没有完全的肿瘤抑制作用；记忆性CTL的完全抑瘤活性需要抗原特异性TH细胞的辅助。结论机体对肿瘤的长期完全杀伤作用不但需要肿瘤特异性抗原诱导产生的记忆性CTL，而且需要特异性TH细胞的辅助，肿瘤疫苗的设计必须包括特异性的CTL表位多肽和辅助性TH细胞多肽。     

Dysfunction of murine dendritic cells induced by incubation with tumor cells

In vivo studies showed that dendritic cell （DC） dysfunction occurred in tumor microenvironment. As tumors were composed of many kinds of cells, the direct effects of tumor cells on immature DCs （imDCs） are needed for further studies in vitro. In the present study, bone marrow-derived imDCs were incubated with lymphoma, hepatoma and menaloma cells in vitro and surface molecules in imDCs were determined by flow cytometry. Then, imDCs incubated with tumor cells or control imDCs were further pulsed with tumor lysates and then incubated with splenocytes to perform mixed lymphocyte reaction. The DC-dependent tumor antigen-specific T cell proliferation, and IL-12 secretion were determined by flow cytometry, and enzyme-linked immunosorbent assay respectively. Finally, the DC-dependent tumor-associated antigen-specific CTL was determined by enzyme-linked immunospot assay. The results showed that tumor cell-DC incubation down-regulated the surface molecules in imDCs, such as CD80, CD54, CDIIb, CDIIa and MHC class II molecules. The abilities of DC-dependent antigen-specific T cell proliferation and IL-12 secretion were also decreased by tumor cell incubation in vitro. Most importantly, the ability for antigenic-specific CTL priming of DCs was also decreased by incubation with tumor cells. In the present in vitro study demonstrated that the defective abilities of DCs induced by tumor cell co-incubation and the co-incubation system might be useful for future study of tumor-immune cells direct interaction and for drug screen of immune-modulation. Cellular ＆ Molecular Immunology.     

FastDC derived from human monocytes within 48 h effectively prime tumor antigen-specific cytotoxic T cells

Previously, we have shown that dendritic cells (DCs) with full T-cell stimulatory capacity can be derived from human monocytes after 48 h of in vitro culture (FastDC). Compared to a standard 7-day protocol, this new strategy not only reduces the time span and the amount of recombinant cytokines required, but may also resemble DC development in vivo more closely. Using a melanoma antigen model, we show here that FastDC prime CTL responses against tumor antigens as effectively as standard monocyte-derived DCs (moDCs). FastDC and moDCs derived from monocytes of HLA-A2(+) donors were loaded with the melanoma-associated, HLA-A(*)0201-restricted peptide Melan-A and cocultured with autologous CD3(+) T cells. After two weekly restimulations with freshly prepared, peptide-loaded FastDC or moDCs, binding of CD8(+) T cells to fluorescently labeled MHC-I/Melan-A-peptide complexes and intracellular cytokine staining revealed that the two DC preparations had an equal capacity to prime Melan-A-specific, IFN-gamma producing CD8(+) T cells. CTLs derived from cocultures with FastDC lysed Melan-A-loaded T2 cells even more effectively than CTLs primed by moDCs. Comparative analysis also revealed that FastDC possess an equal capacity to migrate in response to the chemokine receptor CCR-7 ligand 6Ckine. Importantly, DCs can be generated with higher yield and purity using the FastDC-protocol. The reliability and efficacy of this new strategy for DC development from monocytes may facilitate clinical investigation of DC-based tumor immunotherapy.     

Chemokine receptor trafficking and viral replication

Summary: Chemokines and chemokine receptors have emerged as crucial factors controlling the development and function of leukocytes. Recent studies have indicated that, in addition to these essential roles, both chemokines and chemokine receptors play critical roles in viral infection and replication. Not only are chemokine receptors key components of the receptor/fusion complexes of primate immunodeficiency viruses, hut chemokines can also influence virus entry and infection. Many viruses, in particular herpesviruses, encode chemokines and chemokine receptors that influence the replication of both the parent virus and other unrelated viruses. The cell surface expression of the chemokine receptors is regulated through their interaction with membrane trafficking pathways, ligands induce receptor internalization and downmodulation through endocytosis, and recycling is regulated within endosomes. Pan of the mechanism through which chemokines protect cells from HIV infection is through ligand-induced internalization of the specific chemokine receptor co-receptors. In addition, mechanisms may exist to regulate the trafficking of newly synthesized receptors to the cell surface. Here we discuss aspects of the mechanisms through which chemokine receptors interact with membrane-trafficking pathways and the influence of these interactions on viral replication.     

Differential migration behavior and chemokine production by myeloid and plasmacytoid dendritic cells

The existence of dendritic cell (DC) subsets is firmly established, but their trafficking properties are still largely unknown. We have indicated that myeloid dendritic cells (M-DCs) and plasmacytoid dendritic cells (P-DCs) isolated from human blood differ widely in the capacity to migrate to chemotactic stimuli. The pattern of chemokine receptors expressed ex vivo by both subsets is similar, but P-DCs display, compared with M-DCs, higher levels of CC chemokine receptor (CCR)5, CCR7, and CXCR3. Intriguingly, most chemokine receptors of P-DCs, in particular those specific for inflammatory chemokines and classical chemotactic agonists, are not functional in circulating cells. Following maturation induced by cluster designation (CD)40 ligation, the receptors for inflammatory chemokines are downregulated and CCR7 on P-DCs becomes coupled to migration. The drastically impaired capacity of blood P-DCs to migrate in response to inflammatory chemotactic signals contrasts with the response to lymph node-homing chemokines, indicating a propensity to migrate to secondary lymphoid organs rather than to sites of inflammation. The distinct migration behavior of DC subsets is accompanied by a different profile of chemokine production. In contrast to the high production by M-DCs, the homeostatic CC chemokine ligand (CCL)17/ thymus- and activation-regulated chemokine (TARC) is not produced by PDCs in response to any stimulus tested and their production of CCL22/MDC is minimal, if any, compared with M-DCs. Thus, stimulated M-DCs, but not P-DCs, are able to produce high levels of chemokines recruiting T-helper 2 cells (Th2) and T-regulatory cells. Conversely, the proinflammatory chemokine CCL3/macrophage inflammatory protein (MIP)-1 is predominantly produced by P-DCs. Therefore, P-DCs appear to produce preferentially proinflammatory chemokines, but to respond selectively to homeostatic ones, whereas the reverse is true for M-DCs, highlighting not only the different migratory properties of these DC subsets, but also their capacity to recruit different cell types at inflammation sites.     

G protein-coupled receptors in natural killer cells

Natural killer (NK) cells are capable of killing tumor as well as virally infected cells. How these cells migrate toward the infected sites in the body is not completely understood. Chemokine receptors that belong to the heptahelical family of receptors and characteristically bind heterotrimeric G proteins are present in most NK cells. Recent results showed that resting NK cells highly express constitutive chemokine receptors (CCR4, CCR7, CXCR4, and CX(3)CR1) with low expression of a limited repertoire of inflammatory chemokine receptors (CCR1 and CXCR3). However, only a subset of these cells expressing the CD56(dim) and adhesion molecule(high) phenotype is capable of in vivo binding to vascular endothelium. Under pathological conditions where inflammatory cytokines are present, these cells are induced to express inflammatory chemokine receptors. Resting as well as activated NK cells also express receptors for another member of the heptahelical family of receptors that bind phosphorylated or glycosylated lysolipids. These include sphingosine 1-phosphate (S1P)(1), S1P(4), and S1P(5), the receptors for S1P; lysophosphatidic acid (LPA)(1), LPA(2), and LPA(3), the receptors for LPA; and T cell death-associated gene 8, the receptor for psychosine. Similar to chemokines, S1P, LPA, and psychosine induce the chemotaxis of NK cells through heterotrimeric G proteins. However, in contrast to chemokines, which enhance the cytotoxicity of NK cells, lysolipids inhibit this function. We hope that gaining knowledge regarding the distribution of activated NK cells toward the sites of tumor growth or virally infected sites will give an advantage in designing strategies using these cells as tools for the prevention and treatment of immunodeficiencies.     

Multiple copies of a tumor epitope in a recombinant hepatitis B surface antigen (HBsAg) vaccine enhance CTL responses, but not tumor protection

We propose the replacement of endogenous epitopes with foreign epitopes to exploit the highly immunogenic hepatitis B surface antigen (HBsAg) as a vaccine vector to elicit disease-protective cytotoxic T-lymphocyte (CTL) responses. Locations were defined within the HBsAg gene where replacements of DNA encoding HBsAg epitopes may be made to generate functional recombinant (r) HBsAg DNA vaccines. We demonstrate that rHBsAg DNA vaccines encoding multiple copies of a model tumor epitope from human papillomavirus (HPV) elicit enhanced CTL responses compared to rHBsAg DNA vaccines encoding a single copy. We show that rHBsAg DNA vaccines elicit a marked prophylactic and long-lived therapeutic protection against epitope expressing tumor, although protective efficacy was not improved by increasing the number of copies of the tumor epitope DNA. These results demonstrate the efficacy of HBsAg as a vector for the delivery of foreign CTL epitopes using the epitope replacement strategy, and have implications for rHBsAg vaccine design. The results also have implications for the derivation of a therapeutic vaccine for HPV-associated squamous carcinoma.