Cellular & Molecular Immunology (2013) 10, 303–310 ß 2013 CSI and USTC. All rights reserved 1672-7681/13 $32.00 www.nature.com/cmi

REVIEW

CCL5 as a potential immunotherapeutic target in triple-negative breast cancer

Dandan Lv1, Yan Zhang2, Ha-Jeong Kim2, Lixing Zhang1 and Xiaojing Ma1,2

Breast cancer (BC) is a leading cause of mortality among women in the world. To date, a number of molecules have been established as disease status indicators and therapeutic targets. The best known among them are estrogen receptor-a (ER-a), progesterone receptor (PR) and HER-2/neu. About 15%–20% BC patients do not respond effectively to therapies targeting these classes of tumor-promoting factors. Thus, additional targets are strongly and urgently sought after in therapy for human BCs negative for ER, PR and HER-2, the so-called triple-negative BC (TNBC). Recent clinical work has revealed that CC ligand 5 (CCL5) is strongly associated with the progression of BC, particularly TNBC. How CCL5 contributes to the development of TNBC is not well understood. Experimental animal studies have begun to address the mechanistic issue. In this article, we will review the clinical and laboratory work in this area that has led to our own hypothesis that targeting CCL5 in TNBCs will have favorable therapeutic outcomes with minimal adverse impact on the general physiology. Cellular & Molecular Immunology (2013) 10, 303–310; doi:10.1038/cmi.2012.69; published online 4 February 2013

Keywords: triple negative breast cancer; CCL5; myeloid derived suppressor cell; immunotherapy

CCL5 IN IMMUNITY AND IN INFLAMMATORY DISEASES nophils and . In collaboration with certain are small soluble that act as molecular that are released by T cells such as IL-2 and IFN-c, CCL5 also signals to induce cellular migration during inflammation. induces the activation and proliferation of particular natural- Chemokines and chemokine receptors are expressed by many killer cells to generate CC chemokine-activated killer cells.7 types of cancers cells. Locally produced chemokines can induce CCL5 produced by CD81 T cells and other immune cells has inflammatory tumor-infiltrating cells, facilitate angiogenesis, been shown to inhibit HIV entry into target cells.8 The activities promote growth or survival, and stimulate tumor cell meta- of CCL5 are mediated through its binding to CCR1, CCR3 and stasis.1–3 CCR5.9 CC chemokine ligand 5 (CCL5), also known as RANTES Genetic evidence exists that links CCL5 with numerous (Regulated upon Activation, Normal T-cell Expressed, and human diseases, such as coronary artery disease,10 multiple Secreted), belongs to the CC chemokine family whose mem- sclerosis,11 rheumatoid arthritis,12,13 asthma and atopy,14–17 bers include monocyte chemoattractant (MCP)-1, atopic eczema/dermatitis syndrome, atopic dermatitis,18 hep- MCP-2, MCP-3, I-309, macrophage inhibitory protein-1a atitis C virus infection,19,20 heart graft rejection21 and systemic and macrophage inhibitory protein-1b. CCL5 was first iden- lupus erythematosus.22,23 tified in a search for that were expressed differen- tially in activated T cells but not in B cells.4 In T lymphocytes, CCL5 AND HUMAN BREAST CANCER (BC) CCL5 expression is regulated by Kruppel-like factor 13.4–6 The human CCL5 is located on the long arm of chro- CCL5 is also expressed in macrophages, platelets, synovial mosome 17,24 the same area Her2/neu is encoded and an area fibroblasts, tubular epithelium and certain types of tumor cells. amplified in 30% BC patients.25 Tumor-derived CCL5 is CCL5 plays an active role in recruiting a variety of leukocytes detected in many clinical specimens of breast and cervical can- into inflammatory sites including T cells, macrophages, eosi- cers; greater plasma levels in patients with progressive and 1Sheng Yushou Center of Cell Biology and Immunology, School of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai, China and 2Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY, USA Correspondence: Dr XJ Ma, Sheng Yushou Center of Cell Biology and Immunology, School of Life Science and Biotechnology, 800 Dongchuan Road, Shanghai Jiaotong University, Shanghai 20040, China E-mail: [email protected] or E-mail: [email protected] Received: 30 September 2012; Revised 19 November 2012; accepted: 20 November 2012 CCL5 in triple-negative breast cancer DD Lv et al 304

more advanced disease than those in remission,26–28 and it tumor development induced by Wnt-1 expression is facilitated constitutes a prominent part of a poor prognosis signature by other genetic lesions such as inactivation of p53 and over- of inflammatory BC.29,30 An analysis of large core needle expression of Fgf-3.37 biopsies of 113 invasive breast carcinomas revealed that the concentration of CCL5 was significantly higher in the groups TRIPLE NEGATIVE BC of patients with axillary lymph node metastasis than those ER-a-, PR- and HER-2/neu-negative BC, or triple-negative BC without.31 Most BC-derived cell lines spontaneously produce (TNBC), is a highly malignant disease that traditionally lacks a large amounts of CCL5, and the incidence of CCL5 expression clearly definable therapeutic target. It was originally described detected in tissue sections of breast carcinomas is considerably by Perou et al. as distinct molecular subgroups of BCs differ- higher than in normal duct epithelial cells or in those of benign entiated by their profiles associated with pro- breast lumps.32 CCL5 expression was speculated to be indi- gnosis. These subgroups comprised of a normal breast–like cative of an ongoing, but as yet undetectable, malignant pro- group, the basal epithelial-like group, an ERBB2-overexpres- cess.32 A Cox proportional hazard model-based univariate sing group and the ER-positive luminal A and B groups.38,39 analysis of 142 BC patients indicated little predictive value of Based on their global gene expression profiles, about 70% of CCL5 for disease progression in stage I BC patients. In contrast, breast tumors identified as triple negative by immunohisto- the expression of CCL5, the absence of estrogen receptor (ER)- chemistry belong to the basal-like group of BCs.40 Inte- a and the lack of progesterone receptor (PR) expression in restingly, a large proportion of BCs arising in women with stage II patients increased significantly the risk for disease germline BRCA1 mutations exhibit the triple-negative pheno- development. Conversely, the combinations of CCL5-nega- type and also fall within the basal-like group.41 It is now a tive/ER-a-positive and CCL5-negative/PR-positive in the stage well-accepted notion that polyADP-ribose inhibition within II group as a whole were strongly correlative with an improved BRCA-deficient cancer cells can effective block the residual prognosis.33 DNA repair machinery, resulting in synthetic lethality.42 Furthermore, concomitant expression of the CCL5 and MCP-1 was observed in advanced human BC,34 suggesting 4T1, AN INFLAMMATORY MURINE TNBC MODEL tumor-promoting interactions between these two chemokines. A very commonly used immunocompetent, syngeneic and tri- By immunohistochemistry analysis for the expression of CCL2, ple negative mouse model of mammary tumor development is CCL5, IL-1b and TNF-a, Soria et al. examined four groups of the 4T1 cell line, which was first isolated from a single sponta- patients diagnosed with (i) benign breast disorders; (ii) ductal neously arising mammary tumor from a BALB/cfC3H mouse carcinoma in situ; (iii) invasive ducal carcinoma (IDC) without (murine mammary tumor virus).43 4T1 is an excellent model relapse; and (iv) IDC with relapse, respectively. The analyses system for BC research due to the oncologically and immuno- revealed significant associations between CCL2 and CCL5 and logically well-characterized tumor development. The 4T1 between IL-1b and TNF-a, respectively, in the tumor cells from tumor closely mimics human BC in its physical location, pro- ductal carcinoma in situ and IDC-without-relapse patients. liferative and metastatic characteristics.44 The growth and The expression of CCL2 and CCL5 in the IDC-with-relapse metastatic properties of 4T1 cells closely resemble stage IV group was associated with further elevated expression of IL- BC,45 and it is a TNBC.44 4T1 tumor instinctively migrates 1b and TNF-a.35 primarily through a hematogenous route to many distant In human breast tumor cells, CCL5-containing vesicles on organs including the lung, heart, bone, brain and liver.46 4T1 microtubules traffic from the endoplasmic reticulum to the is also poorly immunogenic in that only slight delays in tumor post-Golgi stage before CCL5 is released, and this process is growth against tumor challenge is attained via immunization controlled by the stiffness of the actin cytoskeleton in a manner with irradiated 4T1 cells, inadequate to protect the host.47 dependent on the (43)TRKN(46) sequence of the 40 s loop of Immune suppression in the 4T1 model has been attributed in CCL5.36 part to a signal transduction and transcription 6-dependent inhibition of the development of tumor-specific CD81 cyto- CCL5 IN EXPERIMENTAL MOUSE MAMMARY TUMORS toxic T lymphocyte activity.48 Nevertheless, 4T1 are capable of The mouse CCL5 is 68-amino acid long and highly basic, shares stimulating an immune response of the host because it has been 85% homology with human CCL5. Most human BC studies in shown that effective anti-tumor immune responses can be vivo are conducted in immunodeficient mice such as SCID induced by dendritic cell mixed with irradiated tumor cells (severe combined immunodeficiency) with human BC cell as a source of uncharacterized tumor antigens.49 In particular, lines such as MCF7, Hs578T, MDA-MB-231 and T47D. substantial reduction in tumor size and in spontaneous Murine mammary carcinoma can be induced in several ways, metastases in the lungs of 4T1 tumor-bearing mice as well as usually by overexpression of oncogenes such as Wnt-1, first prolonged survival time was accomplished by IL-12 administra- identified in mouse mammary tumors as a protooncogene tion.50,51 These effects were accompanied by increased natural activated by insertion of the mammary tumor virus genome. killer activity and enhanced levels of IFN-c in tumor-draining The Wnt-1-mediated mitogenic effect is not dependent on lymph node cells obtained from 4T1 tumor-bearing mice estrogen stimulation, evidenced by tumor formation in ER- treated with IL-12.50 Due to the unique characteristics of 4T1 a-null mice, albeit after an increased latency.37 Mammary tumor, this model has been used to investigate important

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14000 progression of murine mammary carcinoma.57 Met-CCL5, a 12000 dominant negative mutant of CCL5, is able to inhibit experi- mental breast tumor growth in vivo.58 10000 Consistent with its tumor-promoting role in BC, CCL5, 8000 along with CCL2 (also called MCP-1), has been shown to facil- 6000 itate many types of tumor-stimulating cross-talks between the tumor and cells of the surrounding microenvironment. CCL5

Luciferase Units 4000 can cause changes in the composition of the different leukocyte 2000 cell types at the tumor site by accumulating harmful tumor- 0 associated macrophages and by constraining potential anti- CCL5 CCL5- IL-12p40 tumor activities. CCL5 expressed by osteoblasts and NFκB mutant mesenchymal stem cells of the plays Figure 1 Spontaneous ccl5 gene transcription in 4T1 cells. 4T1 tumor a role in breast metastatic processes. CCL5 acts directly on cells (107) were electroporated with 10 mg murine ccl5 promoter– tumor cells to promote their promalignancy phenotype, by reporter construct (2979 to 18),61 or its NF-kB mutant (a 3-base- stimulating their migratory and invasion-related activities.59 substitution at 281 to 279. Also see Figure 3 below), or the IL-12 p40 promoter construct, which is expressed in a highly cell-type spe- TRANSCRIPTIONAL REGULATION OF CCL5 GENE 62 cific manner. Each electroporated sample was split into three equal EXPRESSION IN MAMMARY TUMOR CELLS wells as triplicates. Cells were harvested 48 h later. Cell lysates were Previously, it was shown that 4T1 tumor cells constitutively used for luciferase activity measurement using a luminometer. Total 60 protein content in each sample was used to normalize the data variation produce high levels of CCL2 and CCL5. The high level of due to cell number differences. Data represent triplicate reading for CCL5 may play an important role in the tumor’s growth, meta- each sample with standard deviation. CCL5, CC chemokine ligand 5. stasis and resistance to host immunosurveillance.57 To under- stand the molecular basis for the high spontaneous expression issues such as immunotherapy,52 metastasis,53 anti-angiogen- of CCL5 at the transcriptional level, we performed experiments esis therapy54 and multiple chemotherapy treatments.55,56 in which we transfected a mouse ccl5 gene promoter linked to The 4T1 murine mammary carcinoma cell line expresses the firefly luciferase reporter61 into 4T1 cells by electropora- different levels of CCL5.57 In this model, tumor-derived tion, and measured luciferase activity from the wild type ccl5 CCL5 is able to stimulate the expression of matrix metallopro- promoter and the ccl5 promoter in which the critical NF-kB 61 teinase-9, which can facilitate tumor angiogenesis and hereby, site was mutated by base-substitution. As shown in Figure 1, promote growth in vivo. Tumor-derived CCL5 has also been the wild-type ccl5 promoter was robustly active in 4T1 cells shown to impede anti-tumor T-cell responses and heighten the without any stimulation. This activity was greatly diminished when the NF-kB site was mutated. The IL-12p40 promoter, which is active only in myeloid cells following activation by 62 Antibody lg p50 p65 c-Relp52 pathogens, was totally inactive in 4T1 cells, demonstrating Competitorwt mt the selectivity of ccl5 gene activation in 4T1 tumor cells. These NE M4T data suggest that the ccl5 gene is constitutively and highly expressed due to its transcriptional activation by a mechanism that is critically dependent on NF-kB. Importantly and relevant to this study, NF-kB activation is a hallmark of dysregulated cell growth, malignant transformation, and resistance to apoptosis.63

Spontaneous high NF-kB-binding activity in 4T1 tumor cells Consistent with the total dependence of the ccl5 gene transcrip- Free probe tion on the critical NF-kB element in its promoter, spontan- 1198765432 10 11 2eous and sequence-specific nuclear binding activity of NF-kB Wt -99 to the cognate ccl5 gene promoter between 299 and 267 was -99Mutant observed (Figure 2). NF-kB binding to the ccl5 gene promoter was observed only in 4T1 cells (lane 3), but not in resting Figure 2 Spontaneous NF-kB-binding activity in 4T1 tumor cells. primary macrophages (lane 2). The binding was competed Nuclear extracts (NE) were isolated from 4T1 cells (4T) and primary off efficiently with wild-type ‘cold’ probe (lane 5), but not by mouse macrophages (M). Five mg of NE were mixed with the wild type the NF-kB mutant probe (lane 6). It was specifically ‘super- (wt) or mutant (mt) NF-kB probe (their sequences presented with the critical core sequence underlined). Various competitors and NF-kB shifted’ by antibodies against NF-kB p50 (lane 9) and p65 (lane antibodies and control Ig were used to assess the binding specificity 10), but not by those against c-Rel (lane 11), p52 (lane 12) or and selectivity. The complexes were resolved on a 6% native polyacry- control IgG (lane 8). These results suggest that spontaneously lamide gel. active NF-kB activity in the nucleus of 4T1 cells, composed

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ab 300% 100% 250% 80% 200% 60% 150% 40% 100%

50% 20% Relative promoter activity 0% 0%

Effector control p65 Effector control lκBα-M

R:E molar ratio 1:11:2 1:4 R:E molar ratio 1:1 1:2 1:4

Figure 3 Role of NF-kBinccl5 gene transcription in 4T1 cells. 4T1 tumor cells (107) were electroporated with the murine ccl5 promoter–reporter construct, together with an expression vector for NF-kB p65 (a), or an IkBa mutant (b), at increasing amounts in terms of molar ratios of reporter (R) to effector (E). Cells were harvested 48 h later. Cell lysates were used for luciferase activity measurement. Results are expressed as relative activity to that of ccl5 promoter alone without the effector (p65 or IkBa-M).

mainly of p50 and p65, may be responsible for the constitutive Ras-independent, phosphoinositide-3 kinase-dependent, c- expression of CCL5. Jun N-terminal kinase-dependent phosphorylation of c-Jun To support this hypothesis, we introduced NF-kB p65 into in 4T1 cells results in binding of an AP-1 c-Jun homodimer 4T1 cells by electroporation and measured ccl5 promoter activ- to the promoter.45 We tested if AP-1 was ity. As shown in Figure 3a, p65 expression was able to further involved in driving ccl5 gene transcription in 4T1 cells by enhance ccl5 transcription. Conversely, when we introduced a using a dominant negative mutant of AP-1 called A-Fos. A- constitutively active form of IkBa, which acts as a dominant Fos was engineered on the backbone of the Fos leucine zip- negative against the endogenous IkBa,64 it inhibited ccl5 gene per to which an acidic, amphipathic protein sequence was transcription (Figure 3b). It is noteworthy that the IkBa added at the N terminus. This acidic sequence substitutes the mutant did not completely inhibit ccl5 gene transcription even normal basic region critical for DNA binding. The hetero- at very high concentrations. dimeric coiled-coil structure formed between the acidic extension and the Jun basic region dramatically stabilizes Additional transcription factors may also contribute to ccl5 the complex and prevents the latter region from binding transcription to DNA.65 In addition, several studies have demonstrated The observation that the IkBa mutant could not completely that agents that increase intracellular cyclic AMP inhibit inhibit ccl5 gene transcription (Figure 3b) suggests that addi- the secretion of CCL5 induced by TNF-a66,67 via aCRE- tional transcription factors may be involved through the dependent and AP-1-independent pathway.68 Thus, we used NF-kB site (Figure 1). It has been demonstrated that a a dominant negative mutant of cyclic AMP response element binding protein (CREB), called A-CREB. A-CREB was 200% assembled by melding the N-terminus of the CREB leucine 180% zipper domain with a constructed acidic amphipathic exten- 160% 140% sion. A coiled-coil extension of the leucine zipper is formed 120% when the acidic extension of A-CREB interacts with the basic 100% region of the wild type CREB, rendering the latter domain 80% 69 60% unable to bind DNA. As shown in Figure 4, inhibiting AP- 40% 1 with A-fos resulted in a strong decrease of ccl5 transcrip-

Relative promoter activity 20% tion, while inhibiting CREB with A-CREB had the reverse 0% Vector IKBa-M A-fos A-CREB IKBa-M+ IKBa-M+ effect, suggesting that the endogenous AP-1 may be an A-fos A-CREB important transcriptional activator of ccl5, while CREB may be a repressor. Combining IkBa-M and A-Fos did Figure 4 Role of AP-1 and CREB in ccl5 gene transcription in 4T1 cells not result in greater inhibition of ccl5 transcription than 7 4T1 tumor cells (10 ) were electroporated with the murine ccl5 pro- either effector alone, suggesting that the NF-kB and AP-1 moter–reporter construct, together with an expression vectors for IkBa mutant, A-Fos, and A-CREB at the reporter : effector molar ratio of 1 : 1. pathways operate independently without interaction. Para- Cells were harvested 48 h later. Cell lysates were used for luciferase doxically, when IkBa-M and A-CREB were used together, activity measurement. Results are expressed as relative activity to that the stimulatory effect of A-CREB on ccl5 gene transcription of ccl5 promoter alone without the effectors. CREB, cyclic AMP res- was abrogated, suggesting that CREB may inhibit ccl5 tran- ponse element binding protein. scription in an NF-kB-dependent manner.

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The data shown in Figure 4 suggests that AP-1 may be an impaired capacity to suppress cytotoxicity of CD81 T cells. important transcriptional activator for CCL5 expression. Moreover, tumor-infiltrating MDSCs show increased CCL5 Consistent with this notion, we have observed strong and spon- expression during tumor progression at a greater level than taneous AP-1 binding activity at this site (data not shown). the tumor cells. These properties of CCL5 with respect to Taken together, NF-kB and AP-1 collectively contribute to MDSCs have also been observed in two mouse models of spon- the robust ccl5 gene transcription associated with the properties taneously induced mammary tumors. of 4T1 tumor. MDSCs represent a heterogeneous population of cells com- prised of myeloid progenitors and immature myeloid cells that CCL5 AND OTHER CANCERS contribute to negative regulation of immune responses to A number of other cancer types have been shown to express malignant growth.81 Previous studies have shown in various CCL5,70–74 induce inflammatory cell migration into the tumor mouse models that depletion of MDSCs from tumors or spleens site71,73,74 and enhance tumor growth.70 There is evidence that restores anti-tumor T-cell responses.82–87 The accumulation of inflammatory chemokines including CCL5 expressed by pro- MDSCs is influenced by disturbances of homeostasis state cells may act directly on the growth and survival of hor- in pathological conditions, which stimulate myelopoiesis and mone-driven prostate cancer cells.75 In a Japanese study, it was inhibit the differentiation of mature myeloid cells.81 Recently, found that serum CCL5 concentration was significantly elevated MDSCs have been defined more precisely in mice by two epi- in the ovarian cancer patients compared to the benign ovarian topes recognized by the Gr-1 antibody: Ly6G and Ly6C. cyst patients as controls correlating with the stage of disease and Normally, CD11b1Ly6G2Ly6Chi MDSCs have monocytic the extent of residual tumor mass.76 Mantle cell lymphomas phenotype, whereas CD11b1Ly6G1Ly6Clow MDSCs are of gra- acquire increased expression of CCL4, CCL5 and 4-1BB-L, nulocytic phenotype.81 which are implicated in cell survival.77 CCL5 expression is a In the 4T1 model, our studies further indicate that there is predictor of survival in stage I lung adenocarcinoma.78 Thus, intimate cooperation between tumor-derived macrophage col- CCL5 may well represent a cancer-promoting factor in a broad ony-stimulating factor and granulocyte-macrophage colony- sense as a result of chronic inflammation caused by infection or stimulating factor with CCL5 in the development of immuno- other types of stresses. suppressive MDSCs that promotes tumor expansion. Antibody-mediated, systemic blockade of CCL5 inhibits tumor TUMOR GROWTH INDEPENDENT OF TUMOR-DERIVED progression and strongly enhances the efficacy of vaccination CCL5 against poorly immunogenic 4T1 tumors. Importantly, CCL5 Recent experimental work has indicated that the cellular source helps maintain the immunosuppressive capacity of human of CCL5 expression may not be as simple and straightforward MDSCs. as people originally thought. One study investigated the role of Thus, our study has uncovered a highly novel and important CCL5 produced by tumor cells in promoting growth or meta- chemokine-independent activity of CCL5. Since immune sup- stasis of BC. Two murine mammary tumor models were used. pression plays a crucial role in promoting tumor progression The 4T1 model spontaneously expresses CCL5 and is meta- and failure of cancer vaccines, among which MDSCs are the static, and the 168 tumor does not intrinsically produce major immunosuppressive effectors, different strategies have CCL5 and is not metastatic. CCL5 expression in the 4T1 tumor been proposed to overcome MDSC-mediated immuno- was silenced via RNA interference, while the 168 tumor was suppression. Our study also suggests that targeting CCL5 in engineered to stably express a CCL5 transgene. In both models, TNBC could decrease the immunosuppression activity of no correlation was found of tumor-derived CCL5 expression MDSCs, improve vaccine efficacy against poorly immunogenic with MHC expression, growth rate or metastatic ability of the tumors and reduce BC progression and metastasis. tumors.79,80 These paradoxical results imply that tumor- derived CCL5 expression cannot account for the highly CCL2 AND CXCL8 IN TNBC aggressive nature of the 4T1 tumor. It should be pointed out that CCL2 (also called MCP-1), like CCL5, has been shown in many studies to play an important HEMATOPOIETIC CCL5 AND MYELOID-DERIVED role as well in ER-a-negative breast cancer and TNBC.88 SUPPRESSOR CELLS However, its activity as a regulator of MDSCs has not been Our group has recently obtained data showing that CCL5-defi- explored. In addition, several CXC chemokines are believed cient mice, which develop normally with phenotypically intact to play significant roles in TNBC formation and progression. immune compartments, are nonetheless resistant to advanced For example, CXCL8 (IL-8) expression is negatively linked to 4T1 mammary tumor growth. Bone marrow chimera and ER-a status of breast cancer and CXCL8 expression is asso- adoptive transfer experiments demonstrate that hematopoieti- ciated with a higher invasiveness of cancer cells.89 CXCL8 was cally derived CCL5 plays a dominant role in this phenotype. identified as a key factor involved in breast cancer invasion and Further, the absence of hematopoietic CCL5 causes aberrant angiogenesis.36 However,theroleofCXCL8derivedfromnon- generation of CD11b1Gr-11, myeloid-derived suppressor cells tumor cells in TNBC progression and its role outside the con- (MDSCs) in the bone marrow in response to tumor growth by ventional chemotactic activities have not been established. Thus, accumulating Ly6Chi and Ly6G1, atypical MDSCs with generally speaking, chemokines are double-edged sword. They

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can be instrumental in mobilizing cells in the face of pathogenic 7 Maghazachi AA, Al-Aoukaty A, Schall TJ. CC chemokines induce the generation of killer cells from CD561 cells. Eur J Immunol 1996; 26: infections in order to fight off the invaders. On the other hand, 315–319. they can also be culprits in assisting inflammatory tumors cells 8 Cocchi F, DeVico AL, Garzino-Demo A, Arya SK, Gallo RC, Lusso P. to initiate, propagate and spread. Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV-suppressive factors produced by CD81 T cells. Science 1995; 270: 1811–1815. RATIONALE FOR TARGETING CCL5 IN TNBC THERAPY 9 Pakianathan DR, Kuta EG, Artis DR, Skelton NJ, Hebert CA. Distinct The rationale for targeting CCL5 in TNBC therapy is based on but overlapping epitopes for the interaction of a CC-chemokine with the following considerations: (i) CCL5 appears to be critical for CCR1, CCR3 and CCR5. 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