Expression Of Chemokine Receptor CCR4 Research Articles

SELECTION OF IMMUNOSUPPRESSIVE DRUGS FOR CORD BLOOD DERIVED XENOANTIGEN-STIMULATED TREG

Background: Peripheral blood derived Treg can be cultured and trained as xenoantigen specific Treg which is a promising way for xenotransplantation. However an abnormal quantity and/or quality of regulatory T cells were reported in some autoimmune diseases such as T1D. Thus using an alternative source and selecting Treg-friendly drugs may supply a efficacy immunosuppressive regimen for islet xenotransplantation. This study aims to give experimental evidence regarding the influence of current using immunosuppressive drugs on xenoantigen-stimulated cord blood derived Treg. Methods: Human cord blood derived CD4+CD25+CD127low Treg were cultured with IL-2, rapamycin and irradiated porcine PBMC as xenoantigen stimulation for 2 weeks. The effects of major ISDs as tacrolimus (TAC), mycophenolate (MPA) and new ISD beletacept (BEL) and the mixture of three ISDs (ISDs mix) on xenospecific Treg were test to analysis the cell viability, phenotype, proliferation, function, stability and metabolism. Results: After two weeks induction, xenoantigen stimulated cord blood derived-Tregs (Xn-Treg) have enhanced suppressive capacity in xeno MLR and higher expression of chemokine receptor CCR4, CCR6, CXCR3 and CCR7 compared to polyclonal Treg (Pc-Treg). Further the expression of function molecular CD39, CD73, ICOS, GITR, CTLA-4 and IDO was also upregulated compared to Pc-Treg which confirmed the function of Xn-Treg. Mycophenolate have a negative impact on viability and proliferative capacity of Xn-Treg in a dose-dependent manner. however, BEL and TAC did not affect the viability and proliferative capacity of xenospecific Treg severely. No matter Treg were treated with ISD alone or the mixture of ISDs, the immunosuppressive treatments have no effect on the expression of Treg functional molecules but affected chemokine receptors. From the OCR assay, MPA and ISDs mix significantly increased the maximal respiratory capacity and mitochondrial mass of Xn-Treg compared to the control group. While TAC and BEL did not have any change. For xeno-MLR assays, TAC and MPA did not have any negative effect on the suppressive capacity of Xn-Treg but rather BEL strengthened it at the ratio of responder:Treg 64:1. The results of CBA assay showed when activated CD25-responder cells co-cultured with Treg and ISDs, BEL and Treg have synergetic effect to reduction of IL6, TNF-α, IL17A and IFN-γ which explained why BEL could strengthen the suppressive ability of Treg. Conclusion: Our results suggested xenoantigen-stimulated UCB-Treg show enhanced suppressive capacity in the pig-human MLR and different immune modulatory drugs have different influence on Xn-Treg. We speculate that a low dose combination of ISDs would be better able to support Treg while adequately preventing rejection while minimizing toxicity.

PF268 DECIPHERING THE ABERRANT IMMUNE MICROENVIRONMENT IN ACUTE MYELOID LEUKEMIA DEVELOPMENT AND ITS ROLE IN CHEMOTHERAPY RESPONSE

Background:Comprehensive illustration of phenotypic and functional signatures of abnormal immune microenvironment in acute myeloid leukemia (AML) is lacking.Aims:In this study, we dissected the aberrant immune microenvironment in AML development and its association with chemotherapy response, which may contribute to personalized therapy and provide new insight into novel immunotherapies to augment anti‐leukemia immunity.Methods:Phenotypic and functional analysis of various lymphocytes (eg. CD4+ T cells, CD8+ T cells, NK cells, TNK cells, γδ T cells and B cells) in peripheral blood (PB) and their prognostic implications were performed by multiparameter flow cytometry in 49 patients with AML (before or/and after induction chemotherapy) and 22 healthy controls (HCs).Results:The proportions of CD3+ T cells, NK cells, Th cells, Tc cells and γδ T cells in PB were higher in 25 newly‐diagnosed AML (ND‐AML) patients versus 22 HCs (each P < 0.05). A significant increase in the proportion of CD4+ T (relative to CD3+ T cells), Tfh and Treg cells was also noted, but B cells and TNK cells were similar for ND‐AML and HCs (Fig.1A). As for memory subsets, we found a significantly reduced percentage of naïve subset (CD45RA+CCR7+, relative to CD4+ or CD8+ T cells) in AML at diagnosis. An aberrant unbalance among Th1, Th2, Th17 and Tfh1 cells was also demonstrated in ND‐AML patients. Additionally, the expression of chemokine receptor CCR4, CCR6 and CXCR3 was shown to be down‐regulated. To functionally characterize T cells and NK cells from newly diagnosed AML patients, we assessed several function‐related markers. Further analysis showed a significantly lower expression of CD28 and CD127 in CD4+ or CD8+ T cells, but a higher expression of senescence marker CD57 for ND‐AML versus HCs (Fig.1B). Meanwhile, the homing receptor CD38 and memory marker HLADR demonstrated higher expression in CD8+ T cells in ND‐AML versus HCs. NKG2D expression was found to be significantly lower in NK cells, Vδ1 cells and Vδ2 cells, as well as NKP30 expression in NK cells. The percentage of CD4+ T, CD8+ T, Vδ1 T and Vδ2 T cells expressing inhibitory receptor PD‐1 was significantly higher in AML compared with HCs. Following induction chemotherapy, the immune landscape of AML patients was altered as immune reconstitution occurred. The proportion of CD3+ T cells, CD8+ Tnaïve cells and Th2 cells increased after induction chemotherapy, with the elevated expression of NKG2D in NK and Vδ2 cells, CD28 and CD25 in CD4+ T cells and CD127 in CD8+ T cells. Several phenotypic and functional markers diverged between good responders (GR, n = 19) and refractory or relapsed (RR, n = 14) patients. Good response to induction chemotherapy significantly correlated with downregulation of PD‐1 and CD57 in CD8+ T cells, upregulation of NKG2D in NK cells and Vδ2 cells and increased proportion of CD8+ Tnaïve and Th17 cells (Fig.1C).Summary/Conclusion:Our findings suggest that exhaustion and senescence are the dominant aspects of aberrant immune states in AML at diagnosis. Chemotherapy response and relapse significantly correlate with suppressed immune microenvironment, indicating that immunological markers may be novel prognostic risk factor in acute myeloid leukemia.image

Abstract 3671: Myeloid-derived suppressive cells require education from tumor-evoked Bregs to mediate metastasis

Abstract Myeloid-derived suppressive cells (MDSC) are key metastasis-promoting regulatory cells. They are drastically expanded in WT BALB/c mice with highly aggressive 4T1 cancer and thought to promote metastasis by inactivating antitumor effector immune cells. Surprisingly, despite comparable expansion of MDSC, in B-cell deficient congenic mice 4T1 cancer fails to metastasize to lungs, questioning the importance of MDSC. We found that this is due to the lack of a new subset of tumor-evoked regulatory B cells (tBregs) 1,2 that induces metastasis-promoting regulatory T cells (Tregs) 3. The mechanism of this process is that cancer initiates a chain of metastasis-promoting events, such as converts normal B cells into tBregs utilizing metabolites of 5-lipooxygenase, which in turn use TGFβ to generate FoxP3+ Tregs from non-regulatory CD4+ T cells. The Tregs then inactivate effector NK and CD8+ T cells that kill the metastasizing cancer cells in the lungs. To reconcile these results with the reported importance of MDSC, we speculated that metastasis-promoting activity of MDSC requires tBregs. Here we report that tBregs educate a small subset of MDSC rendering them regulatory. As a result, the ability of 4T1 cancer to metastasize in B-cell KO mice can be restored by the adoptive transfer of the regulatory subset of MDSC from tumor-bearing WT mice. In tumor-bearing B-cell KO mice, however, tBregs are not generated and thus MDSC from these mice cannot support lung metastasis. Overall, these results further underscore the importance of tBregs by uncovering a previously unknown regulatory step that MDSC can only promote metastasis if educated by cancer-induced tBregs. This work was supported by the National Institute on Aging, the Intramural Research Program 1 Olkhanud, P. B. et al. Tumor-evoked regulatory B cells promote breast cancer metastasis by converting resting CD4 T cells to T-regulatory cells. Cancer research 71, 3505-3515, doi:10.1158/0008-5472.CAN-10-4316 (2011). 2 Bodogai, M. et al. Anti-CD20 antibody promotes cancer escape via enrichment of tumor-evoked regulatory B cells expressing low levels of CD20 and CD137L. Cancer research, doi:10.1158/0008-5472.CAN-12-4184 (2013). 3 Olkhanud, P. B. et al. Breast cancer lung metastasis requires expression of chemokine receptor CCR4 and regulatory T cells. Cancer Res. 69, 5996-6004 (2009). Citation Format: Monica Bodogai, Catalina Lee Chang, Arya Biragyn. Myeloid-derived suppressive cells require education from tumor-evoked Bregs to mediate metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3671. doi:10.1158/1538-7445.AM2014-3671

Aberrant expression of chemokine receptor CCR4 in human gastric cancer contributes to tumor‐induced immunosuppression

The chemokine receptor CCR4 is preferentially expressed on certain immune cells and some hematological tumor cells, which play pivotal roles in suppression of host immune response. However, the reasons for the upmodulation of CCR4 and its immune functions in solid tumors remain unclear. Herein, we aimed to determine the expression profiles of CCR4 in gastric cancer cells and its role in regulating antitumor immunity. CCR4 expression was assessed in 63 cases of gastric carcinomas by immunohistochemistry. We found cancer cells in lymphocyte-rich carcinomas more frequently showed moderate to strong positive staining for CCR4 than those in conventional carcinomas (P = 0.041), and also found a positive relationship between expression of CCR4 and tumor necrosis factor-α (P = 0.012). Stimulation of gastric cell lines with various cytokines showed that tumor necrosis factor-α uniquely upmodulated CCR4 expression through activation of nuclear factor-κB. Additional coculture experiments showed the forced expression of CCR4 in SGC-7901 cells caused a significant reduction of γ-interferon and elevation of interleukin-10 secretion in the supernatants from cocultured SGC-7901 cells and PBMCs. In addition, granzyme A production in cancer cell-cocultured CD56(+) natural killer cells was significantly downregulated. Inhibition of the overexpressed CCR4 in cancer cells by an inhibitor of CCR4, compound 39, proved to partly restore the antitumor immunity in respect of the inverse changes in those factors. Our studies suggest that the aberrant expression of CCR4 in human gastric cancer could contribute to tumor-induced immunosuppression. Conceivably, downmodulation of CCR4 expression could be a promising immunotherapy for human gastric cancer.

Immune response deviation and enhanced expression of chemokine receptor CCR4 in TBI patients due to unknown serum factors

Background Severe brain trauma leads to an activation of the immune system. To this date, neither the exact perturbation of the specific immune reaction induced by the traumatic brain injury (TBI), nor the interactions leading to the infiltration of peripheral immune cells into the brain are fully understood. Patients and methods Serum was collected from 17 patients with TBI and a long bone fracture, 24 patients with an isolated long bone fracture and from healthy individuals. The effect of the serum on normal human monocytes and T-lymphocytes was tested in vitro by assessing proliferation and expression of surface markers, chemokine receptors and cytokines. Results Serum collected from patients with a TBI and a long bone fracture increased the expression of the chemokine receptor CCR4 in monocytes when compared to patients with an isolated long bone fracture. Extending this comparison to T-lymphocytes, the serum from TBI patients induced lower proliferation rates and decreased expression of the pro-inflammatory cytokine TNF-α, while simultaneously increasing the secretion of immune-modulatory cytokines (IL-4, IL-10 and TGF-β) ( p < 0.05). Conclusion Patients with a TBI release currently unknown soluble factors into the circulating blood that up regulate expression of chemokine receptor CCR4 in peripheral blood monocytes whilst concurrently inducing expression of immunosuppressive cytokines by activated T-lymphocytes.

Breast Cancer Lung Metastasis Requires Expression of Chemokine Receptor CCR4 and Regulatory T Cells

Cancer metastasis is a leading cause of cancer morbidity and mortality. More needs to be learned about mechanisms that control this process. In particular, the role of chemokine receptors in metastasis remains controversial. Here, using a highly metastatic breast cancer (4T1) model, we show that lung metastasis is a feature of only a proportion of the tumor cells that express CCR4. Moreover, the primary tumor growing in mammary pads activates remotely the expression of TARC/CCL17 and MDC/CCL22 in the lungs. These chemokines acting through CCR4 attract both tumor and immune cells. However, CCR4-mediated chemotaxis was not sufficient to produce metastasis, as tumor cells in the lung were efficiently eliminated by natural killer (NK) cells. Lung metastasis required CCR4(+) regulatory T cells (Treg), which directly killed NK cells using beta-galactoside-binding protein. Thus, strategies that abrogate any part of this process should improve the outcome through activation of effector cells and prevention of tumor cell migration. We confirm this prediction by killing CCR4(+) cells through delivery of TARC-fused toxins or depleting Tregs and preventing lung metastasis.

ORIGINAL ARTICLE: Th1 and Th2 in Human IVF Pregnancy with Allogenic Fetus

Problem Positive obstetric outcome of allogenic in vitro fertilization (IVF) pregnancies makes interesting the subject of additional regulatory mechanisms after oocyte donation.Method of study Eighty eight women: 23 donation of oocytes (DO), 33 IVF, 32 natural conception (NC) were studied in first trimester of pregnancy. Intracellular production of cytokines and chemokine receptors expression were studied by flow cytometry.Results Intracellular production of interferon‐γ (IFN‐γ), interleukin (IL)‐4, tumor necrosis factor‐alpha by CD4 T lymphocytes in DO women was higher than in IVF and NC women. Ratio IFN‐γ/IL‐4 in DO was lower than in IVF. We found higher expression of chemokine receptor CCR4 but not CXCR3 on CD4 T cells in DO compared with IVF and NC. Ratio CXCR3/CCR4 in DO was lower than in NC.Conclusion Hyperactivation of T helper 1 (Th1) and T helper 2 (Th2) by allogenic fetus is specific for DO pregnancy. Preferable activation of Th2 and relative suppression of Th1 chemokine expression reflect additional regulatory counteractive mechanism(s).

Expression of Chemokine Receptor CCR4 and Its Ligands (CCL17 and CCL22) in Murine Contact Hypersensitivity

Chemokine receptor CCR4 and its ligands (CCL17 and CCL22) are important for the recruitment of memory T cells into the skin in various cutaneous immune diseases. However, information on CCR4 and its ligands in contact hypersensitivity is relatively limited. In this study, we investigated the expression of CCR4, CCL17, and CCL22 in a mouse model of contact hypersensitivity to oxazolone. Contact sensitization to oxazolone increased the proportions of memory CD4+ T cells in the draining lymph nodes, spleen, and peripheral blood. Although CCR4+ mRNA and CCR4+ cells were detectable in naive mouse lymph nodes, they significantly increased in the sensitized mice. The majority of CCR4+ cells in both control and sensitized mouse lymph nodes were CD4+ T cells. In the skin of naive mice, the mRNAs for CCR4, CCL17, and CCL22 were detectable, but only CCL17 and CCL22 proteins were constitutively expressed in the skin, particularly in the epidermis. Interestingly, the mRNAs for CCR4 and its two ligands were significantly elevated in the inflamed skin of mice with contact hypersensitivity to oxazolone. Furthermore, a subpopulation of cells that infiltrated the skin was CCR4+ cells. Finally, the expression of CCL17 and CCL22 proteins was significantly enhanced in the epidermis of inflamed skin. Thus, our study provides direct evidence for the presence of CCR4 and its ligands in mouse contact hypersensitivity.

Cutting Edge: Chemokine Receptor CCR4 Is Necessary for Antigen-Driven Cutaneous Accumulation of CD4 T Cells under Physiological Conditions

Dual expression of chemokine receptor CCR4 and E-selectin ligand is characteristic of skin-tropic CD4 T cells from blood, lymphoid organs, and the skin itself. A strong and specific correlation exists among CCR4, its ligand CCL17/TARC, and the cutaneous lymphocyte-homing process. Nevertheless, whether CCR4 function is required for skin-specific trafficking remains an open question, which we address in this study. We developed an Ag-specific, TCR-transgenic, murine CD4 T cell adoptive transfer model that induces a mixed Th1 and Th17 cutaneous response. Within the hosts, both CCR4(+/+) and CCR4(-/-) donor CD4 T cells contribute equally well to the circulating E-selectin ligand(+) pool in response to Ag. However, only CCR4(+/+) donor cells accumulate efficiently within the skin. CCR4(-/-) cells home normally to the peritoneum, showing that they do not have a general defect in lymphocyte trafficking. We conclude that under physiological conditions, CCR4 is a nonredundant, necessary component of skin-specific lymphocyte trafficking.