Tumor-Specific Regulatory T Cells from the Bone Marrow Orchestrate Antitumor Immunity in Breast Cancer.

Endogenous antitumor effector T-cell responses and immune-suppressive regulatory T cells (Treg) critically influence the prognosis of patients with cancer, yet many of the mechanisms of how this occurs remain unresolved. On the basis of an analysis of the function, antigen specificity, and distribution of tumor antigen-reactive T cells and Tregs in patients with breast cancer and transgenic mouse tumor models, we showed that tumor-specific Tregs were selectively activated in the bone marrow (BM) and egressed into the peripheral blood. The BM was constantly depleted of tumor-specific Tregs and was instead a site of increased induction and activity of tumor-reactive effector/memory T cells. Treg egress from the BM was associated with activation-induced expression of peripheral homing receptors such as CCR2. Because breast cancer tissues express the CCR2 ligand CCL2, the activation and egress of tumor antigen-specific Tregs in the BM resulted in the accumulation of Tregs in breast tumor tissue. Such immune compartmentalization and redistribution of T-cell subpopulations between the BM and peripheral tissues were achieved by vaccination with adenoviral vector-encoded TRP-2 tumor antigen in a RET transgenic mouse model of spontaneous malignant melanoma. Thus, the BM simultaneously represented a source of tumor-infiltrating Tregs and a site for the induction of endogenous tumor-specific effector T-cell responses, suggesting that both antitumor immunity and local immune suppression are orchestrated in the BM.

Protein Phosphatase 1α and Cofilin Regulate Nuclear Translocation of NF-κB and Promote Expression of the Anti-Inflammatory Cytokine Interleukin-10 by T Cells.

While several protein serine/threonine kinases control cytokine production by T cells, the roles of serine/threonine phosphatases are largely unexplored. Here, we analyzed the involvement of protein phosphatase 1α (PP1α) in cytokine synthesis following costimulation of primary human T cells. Small interfering RNA (siRNA)-mediated knockdown of PP1α (PP1KD) or expression of a dominant negative PP1α (D95N-PP1) drastically diminished interleukin-10 (IL-10) production. Focusing on a key transcriptional activator of human IL-10, we demonstrate that nuclear translocation of NF-κB was significantly inhibited in PP1KD or D95N-PP1 cells. Interestingly, knockdown of cofilin, a known substrate of PP1 containing a nuclear localization signal, also prevented nuclear accumulation of NF-κB. Expression of a constitutively active nonphosphorylatable S3A-cofilin in D95N-PP1 cells restored nuclear translocation of NF-κB and IL-10 expression. Subpopulation analysis revealed that defective nuclear translocation of NF-κB was most prominent in CD4+ CD45RA- CXCR3- T cells that included IL-10-producing TH2 cells. Together these findings reveal novel functions for PP1α and its substrate cofilin in T cells namely the regulation of the nuclear translocation of NF-κB and promotion of IL-10 production. These data suggest that stimulation of PP1α could limit the overwhelming immune responses seen in chronic inflammatory diseases.

Circulating and Tumor Myeloid-derived Suppressor Cells in Resectable Non-Small Cell Lung Cancer.

RATIONALE: Myeloid-derived suppressor cell (MDSC) expansion has been found to play a role in disease progression in patients with cancer. However, the characteristics of MDSCs in lung cancer are poorly understood. OBJECTIVES: We prospectively investigated MDSCs and inflammatory factors in tumor and peripheral blood samples from patients with resectable non-small cell lung cancer and studied their correlations with the disease prognosis. METHODS: A complex analysis of MDSC subsets and inflammatory mediators was performed using flow cytometry and a Bio-Plex assay. MEASUREMENTS AND MAIN RESULTS: A significant increase in the frequency of circulating monocytic (M)-MDSCs was observed in the patients with non-small cell lung cancer compared with the healthy donors (HDs). Moreover, the frequencies of M- and polymorphonuclear (PMN)-MDSCs were higher in tumors than in the peripheral blood of the same patients. This accumulation was associated with elevated concentrations of inflammatory mediators involved in MDSC migration to and activation in the tumor microenvironment. An analysis of the MDSC immunosuppressive pattern showed increased programmed death-ligand 1 expression on circulating cells from patients compared with HDs. Tumor PMN-MDSCs displayed higher programmed death-ligand 1 expression levels than the same cells in the peripheral blood. The frequency of CCR5 (C-C chemokine receptor 5) expression on circulating M-MDSCs was significantly higher in the patients than in the HDs. Clinical data analysis revealed negative correlations between recurrence-free survival and the frequencies of PMN-MDSCs and CCR5+ M-MDSCs in the circulation but not in tumors. CONCLUSIONS: Our findings suggest that the level of MDSCs in the peripheral blood but not in tumor tissues predicts recurrence after surgery.

Tumor specific regulatory T cells in the bone marrow of breast cancer patients selectively upregulate the emigration receptor S1P1.

Regulatory T cells (Treg) hamper anti-tumor T-cell responses resulting in reduced survival and failure of cancer immunotherapy. Among lymphoid organs, the bone marrow (BM) is a major site of Treg residence and recirculation. However, the process governing the emigration of Treg from BM into the circulation remains elusive. We here show that breast cancer patients harbour reduced Treg frequencies in the BM as compared to healthy individuals or the blood. This was particularly the case for tumor antigen-specific Treg which were quantified by MHCII tumor peptide loaded tetramers. We further demonstrate that decreased Treg distribution in the BM correlated with increased Treg redistribution to tumor tissue, suggesting that TCR triggering induces a translocation of Treg from the BM into tumor tissue. Sphingosine-1-phosphate receptor 1 (S1P1)-which is known to mediate exit of immune cells from lymphoid organs was selectively expressed by tumor antigen-specific BM Treg. S1P1 expression could be induced in Treg by BM-resident antigen-presenting cells (BMAPCs) in conjunction with TCR stimulation, but not by TCR stimulation or BMAPCs alone and triggered the migration of Treg but not conventional T cells (Tcon) to its ligand Sphingosine-1-phosphate (S1P). Interestingly, we detected marked S1P gradients between PB and BM in breast cancer patients but not in healthy individuals. Taken together, our data suggest a role for S1P1 in mediating the selective mobilization of tumor specific Treg from the BM of breast cancer patients and their translocation into tumor tissue.

Macrophage-derived nitric oxide initiates T-cell diapedesis and tumor rejection.

In tumor biology, nitric oxide (NO) is generally regarded as an immunosuppressive molecule that impedes T-cell functions and activation of endothelial cells. Contrasting with this view, we here describe a critical role for NO derived from inducible nitric oxide (iNOS)-expressing tumor macrophages in T-cell infiltration and tumor rejection as shown by iNOS gene deletion, inhibition of iNOS, or NO donors. Specifically, macrophage-derived NO was found to induce on tumor vessels adhesion molecules that were required for T-cell extravasation. Experiments with human endothelial cells revealed a bimodal dose-dependent effect of NO. High doses of NO donors were indeed suppressive but lower, more physiological concentrations, induced adhesion molecules in an NFkB-dependent pathway and preferentially activated transcription of genes involved in lymphocyte diapedesis. iNOS+ macrophages in tumors appear to generate precisely the amount of NO that promotes endothelial activation and T-cell infiltration. These results will be valuable for the development of strategies designed to overcome the paucity of T-cell infiltration into tumors that is a major obstacle in clinical cancer immunotherapy.

Myeloid Cells and Related Chronic Inflammatory Factors as Novel Predictive Markers in Melanoma Treatment with Ipilimumab.

PURPOSE: Immunotherapy with ipilimumab improves the survival of patients with metastatic melanoma. Because only around 20% of patients experience long-term benefit, reliable markers are needed to predict a clinical response. Therefore, we sought to determine if some myeloid cells and related inflammatory mediators could serve as predictive factors for the patients' response to ipilimumab. EXPERIMENTAL DESIGN: We performed an analysis of myeloid cells in the peripheral blood of 59 stage IV melanoma patients before the treatment and at different time points upon the therapy using a clinical laboratory analysis and multicolor flow cytometry. In addition, the production of related inflammatory factors was evaluated by ELISA or Bio-Plex assays. RESULTS: An early increase in eosinophil count during the treatment with ipilimumab was associated with an improved clinical response. In contrast, elevated amounts of monocytic myeloid-derived suppressor cells (moMDSC), neutrophils, and monocytes were found in nonresponders (n = 36) as compared with basal levels and with responding patients (n = 23). Moreover, in nonresponders, moMDSCs produced significantly more nitric oxide, and granulocytic MDSCs expressed higher levels of PD-L1 than these parameters at baseline and in responders, suggesting their enhanced immunosuppressive capacity. Upon the first ipilimumab infusion, nonresponders displayed elevated serum concentrations of S100A8/A9 and HMGB1 that attract and activate MDSCs. CONCLUSIONS: These findings highlight additional mechanisms of ipilimumab effects and suggest levels of eosinophils, MDSCs, as well as related inflammatory factors S100A8/A9 and HMGB1 as novel complex predictive markers for patients who may benefit from the ipilimumab therapy. Clin Cancer Res; 21(24); 5453-9. ©2015 AACR.

von Willebrand factor fibers promote cancer-associated platelet aggregation in malignant melanoma of mice and humans.

Tumor-mediated procoagulatory activity leads to venous thromboembolism and supports metastasis in cancer patients. A prerequisite for metastasis formation is the interaction of cancer cells with endothelial cells (ECs) followed by their extravasation. Although it is known that activation of ECs and the release of the procoagulatory protein von Willebrand factor (VWF) is essential for malignancy, the underlying mechanisms remain poorly understood. We hypothesized that VWF fibers in tumor vessels promote tumor-associated thromboembolism and metastasis. Using in vitro settings, mouse models, and human tumor samples, we showed that melanoma cells activate ECs followed by the luminal release of VWF fibers and platelet aggregation in tumor microvessels. Analysis of human blood samples and tumor tissue revealed that a promoted VWF release combined with a local inhibition of proteolytic activity and protein expression of ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type I repeats 13) accounts for this procoagulatory milieu. Blocking endothelial cell activation by the low-molecular-weight heparin tinzaparin was accompanied by a lack of VWF networks and inhibited tumor progression in a transgenic mouse model. Our findings implicate a mechanism wherein tumor-derived vascular endothelial growth factor-A (VEGF-A) promotes tumor progression and angiogenesis. Thus, targeting EC activation envisions new therapeutic strategies attenuating tumor-related angiogenesis and coagulation.

NF1 loss induces senescence during human melanocyte differentiation in an iPSC-based model.

Neurofibromatosis type 1 (NF1) is a frequent genetic disease leading to the development of Schwann cell-derived neurofibromas or melanocytic lesions called café-au-lait macules (CALMs). The molecular mechanisms involved in CALMs formation remain largely unknown. In this report, we show for the first time pathophysiological mechanisms of abnormal melanocyte differentiation in a human NF1(+/-) -induced pluripotent stem cell (iPSC)-based model. We demonstrate that NF1 patient-derived fibroblasts can be successfully reprogrammed in NF1(+/-) iPSCs with active RAS signaling and that NF1 loss induces senescence during melanocyte differentiation as well as in patient's-derived CALMs, revealing a new role for NF1 in the melanocyte lineage.

A high-throughput RNAi screen for detection of immune-checkpoint molecules that mediate tumor resistance to cytotoxic T lymphocytes.

The success of T cell-based cancer immunotherapy is limited by tumor's resistance against killing by cytotoxic T lymphocytes (CTLs). Tumor-immune resistance is mediated by cell surface ligands that engage immune-inhibitory receptors on T cells. These ligands represent potent targets for therapeutic inhibition. So far, only few immune-suppressive ligands have been identified. We here describe a rapid high-throughput siRNA-based screening approach that allows a comprehensive identification of ligands on human cancer cells that inhibit CTL-mediated tumor cell killing. We exemplarily demonstrate that CCR9, which is expressed in many cancers, exerts strong immune-regulatory effects on T cell responses in multiple tumors. Unlike PDL1, which inhibits TCR signaling, CCR9 regulates STAT signaling in T cells, resulting in reduced T-helper-1 cytokine secretion and reduced cytotoxic capacity. Moreover, inhibition of CCR9 expression on tumor cells facilitated immunotherapy of human tumors by tumor-specific T cells in vivo. Taken together, this method allows a rapid and comprehensive determination of immune-modulatory genes in human tumors which, as an entity, represent the 'immune modulatome' of cancer.

Elevated chronic inflammatory factors and myeloid-derived suppressor cells indicate poor prognosis in advanced melanoma patients.

Chronic inflammation is considered to be one of the hallmarks for tumor initiation and progression. Moreover, a long-term production and accumulation of inflammatory factors lead to a local and systemic immunosuppression associated with cancer progression. However, the correlation between inflammatory mediators, immunosuppressive cells and the clinical outcome of malignant melanoma patients was poorly investigated. In this study, we performed a complex analysis of various inflammatory factors, myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in the peripheral blood of patients suffering from malignant melanoma of different stages. We demonstrated that levels of serum IL-1ß, IFN-γ and CXCL10 were significantly increased in advanced melanoma patients. In addition, these factors were found to be associated with an increased frequency of MDSCs and Tregs as compared to age- and gender-matched healthy donors. Importantly, advanced melanoma patients with signs of progression displayed markedly elevated concentrations of IL-1ß and CXCL10 as compared to patients with stable disease. Moreover, an enrichment of circulating monocytic (Mo)-MDSCs significantly correlated with a decreased progression free survival of these patients. Our data highlight a complex association between circulating inflammatory mediators, Mo-MDSCs and the clinical outcome as well as suggest that their levels in patients with advanced melanoma are of important prognostic value allowing the identification of those with high risk of disease progression.

BRaf and MEK inhibitors differentially regulate cell fate and microenvironment in human hepatocellular carcinoma.

PURPOSE: Small molecule inhibitors of the mitogen-activated protein kinase (MAPK) pathway, such as sorafenib, represent novel treatment options for advanced hepatocellular carcinoma. The aim of our study was to identify downstream targets as biomarker candidates that are directly linked to the oncogenic MAPK pathway in hepatocellular carcinoma and correlate with inhibition of this pathway by multikinase inhibitors. EXPERIMENTAL DESIGN: Hepatocellular carcinoma cell lines and fresh tumor and tumor-free liver tissues from patients with hepatocellular carcinoma were incubated with different BRaf or MEK inhibitors and analyzed for kinase phosphorylation, proliferation, induction of apoptosis, and chemokine secretion. RESULTS: Hepatocellular carcinoma cell lines responded differentially to these inhibitors in a dose-dependent manner, even those targeting the same kinase. Sorafenib inhibited both MEK1 and ERK1/2 phosphorylation at high but increased signaling at low concentrations. Similarly, PLX4720 increased MEK/ERK signaling independently from mutations in BRaf or NRas. MEK inhibitors decreased ERK1/2 phosphorylation in a dose-dependent manner. These signaling characteristics correlated with inhibition of proliferation, induction of apoptosis, and chemokine secretion. Fresh tissues derived from patients diagnosed with primary hepatocellular carcinoma responded to these inhibitors with changes in their microenvironment following the patterns observed in hepatocellular carcinoma cells. CONCLUSIONS: Oncogenic signaling of the MAPK pathway influences hepatocellular carcinoma sensitivity to treatment with BRaf and MEK inhibitors about cell fate independently from mutations in BRaf and NRas. MAPK inhibitors have a strong impact on chemokine secretion as a consequence of interference with oncogenic signaling. Therefore, novel biomarker candidates associated with the hepatocellular carcinoma microenvironment may be developed for prediction and monitoring of treatment response to small molecule inhibitors.

Low-dose irradiation programs macrophage differentiation to an iNOS⁺/M1 phenotype that orchestrates effective T cell immunotherapy.

Inefficient T cell migration is a major limitation of cancer immunotherapy. Targeted activation of the tumor microenvironment may overcome this barrier. We demonstrate that neoadjuvant local low-dose gamma irradiation (LDI) causes normalization of aberrant vasculature and efficient recruitment of tumor-specific T cells in human pancreatic carcinomas and T-cell-mediated tumor rejection and prolonged survival in otherwise immune refractory spontaneous and xenotransplant mouse tumor models. LDI (local or pre-adoptive-transfer) programs the differentiation of iNOS⁺ M1 macrophages that orchestrate CTL recruitment into and killing within solid tumors through iNOS by inducing endothelial activation and the expression of TH1 chemokines and by suppressing the production of angiogenic, immunosuppressive, and tumor growth factors.

Body fluid exosomes promote secretion of inflammatory cytokines in monocytic cells via Toll-like receptor signaling.

Tumor-derived exosomes have been shown to induce various immunomodulatory effects. However, the underlying signaling pathways are poorly understood. Here, we analyzed the effects of ex vivo-derived exosomes on monocytic cell differentiation/activation using THP-1 cells as model. We isolated exosomes from various body fluids such as amniotic fluid, liver cirrhosis ascites, and malignant ascites of ovarian cancer patients. We observed that exosomes were internalized by THP-1 cells and induced the production of IL-1ß, TNF-α, and IL-6. Analysis of the signaling pathways revealed a fast triggering of NFκB and a delayed activation of STAT3. Pharmacologic and antibody-blocking experiments showed that the initial production of IL-6 was instrumental for subsequent activation of STAT3. Importantly, triggering of cell signaling was not a unique property of tumor exosomes but was also observed with exosomes of noncancerous origin. Exosomal signaling was TLR-dependent as the knockdown of Toll-like receptor 2 (TLR2) and TLR4 blocked NFκB and STAT3 activation. Similar results were obtained with TLR-neutralizing antibodies. Exosomes also triggered the release of cytokines from mouse bone marrow-derived dendritic cells or macrophages. This process was MyD88-dependent, further supporting a role of TLR signaling. Our results suggest that exosomes trigger TLR-dependent signaling pathways in monocytic precursor cells but possibly also in other immune cells. This process could be important for the induction of immunosuppressive mechanisms during cancer progression and inflammatory diseases.

Tumor cells in multiple myeloma patients inhibit myeloma-reactive T cells through carcinoembryonic antigen-related cell adhesion molecule-6.

Although functionally competent cytotoxic, T cells are frequently observed in malignant diseases, they possess little ability to react against tumor cells. This phenomenon is particularly apparent in multiple myeloma. We here demonstrate that cytotoxic T cells reacted against myeloma antigens when presented by autologous dendritic cells, but not by myeloma cells. We further show by gene expression profiling and flow cytometry that, similar to many other malignant tumors, freshly isolated myeloma cells expressed several carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) at varying proportions. Binding and crosslinking of CEACAM-6 by cytotoxic T cells inhibited their activation and resulted in T-cell unresponsiveness. Blocking of CEACAM-6 on the surface of myeloma cells by specific monoclonal antibodies or CEACAM-6 gene knock down by short interfering RNA restored T-cell reactivity against malignant plasma cells. These findings suggest that CEACAM-6 plays an important role in the regulation of CD8+ T-cell responses against multiple myeloma; therefore, therapeutic targeting of CEACAM-6 may be a promising strategy to improve myeloma immunotherapy.

Effects of cyclophosphamide and IL-2 on regulatory CD4+ T cell frequency and function in melanoma patients vaccinated with HLA-class I peptides: impact on the antigen-specific T cell response.

The frequency and function of regulatory T cells (Tregs) were studied in stage II-III melanoma patients who were enrolled in a phase II randomized trial of vaccination with HLA-A*0201-modified tumor peptides versus observation. The vaccinated patients received low-dose cyclophosphamide (CTX) and low-dose interleukin-2 (IL-2). Tregs were analyzed in the lymph nodes (LNs) of stage III patients who were undergoing complete lymph node dissection and in peripheral blood mononuclear cells (PBMCs) collected before vaccination and at different time points during the vaccination period. The LNs of the vaccinated patients, which were surgically removed after two rounds of vaccination and one dose of CTX, displayed a low frequency of Tregs and a less immunosuppressive environment compared with those of the untreated patients. The accurate time-course analysis of the PBMCs of patients enrolled in the vaccination arm indicated a limited and transient modulation in the frequencies of Tregs in PBMCs collected after low-dose CTX administration and a strong Treg boost in those PBMCs collected after low-dose IL-2 administration. However, a fraction of the IL-2-boosted Tregs was functionally modulated to a Th-1-like phenotype in the vaccinated patients. Moreover, low-dose IL-2 promoted the concomitant expansion of conventional activated CD4(+) T cells. Despite the amplification of Tregs, IL-2 administration maintained or further increased the number of antigen-specific CD8(+) T cells that were induced by vaccination as demonstrated by the ex vivo human leukocyte antigen-multimer staining and IFN-γ ELISpot assays. Our study suggests that the use of CTX as a Treg modulator should be revised in terms of the administration schedule and of patients who may benefit from this drug treatment. Despite the Treg expansion that was observed in this study, low-dose IL-2 is not detrimental to the functional activities of vaccine-primed CD8(+) T cell effectors when used in the inflammatory environment of vaccination.

Antitumor effect of paclitaxel is mediated by inhibition of myeloid-derived suppressor cells and chronic inflammation in the spontaneous melanoma model.

The antitumor effects of paclitaxel are generally attributed to the suppression of microtubule dynamics resulting in defects in cell division. New data demonstrated that in ultralow noncytotoxic concentrations, paclitaxel modulated in immune cells in vitro the activity of small Rho GTPases, the key regulators of intracellular actin dynamics. However, the immunomodulatory properties of paclitaxel in vivo have not been evaluated. In this study, using the ret transgenic murine melanoma model, which mimics human cutaneous melanoma, we tested effects of ultralow noncytotoxic dose paclitaxel on functions of myeloid-derived suppressor cells (MDSCs), chronic inflammatory mediators, and T cell activities in the tumor microenvironment in vivo. Administration of paclitaxel significantly decreased accumulation and immunosuppressive activities of tumor-infiltrating MDSCs without alterations of the bone marrow hematopoiesis. This was associated with the inhibition of p38 MAPK activity, TNF-α and production, and S100A9 expression in MDSCs. The production of mediators of chronic inflammation in the tumor milieu also was diminished. Importantly, reduced tumor burden and increased animal survival upon paclitaxel application was mediated by the restoration of CD8 T cell effector functions. We suggest that the ability of paclitaxel in a noncytotoxic dose to block the immunosuppressive potential of MDSCs in vivo represents a new therapeutic strategy to downregulate immunosuppression and chronic inflammation in the tumor microenvironment for enhancing the efficacy of concomitant anticancer therapies.

Cyclophosphamide promotes chronic inflammation-dependent immunosuppression and prevents antitumor response in melanoma.

Low-dose cyclophosphamide (CP) therapy induces immunogenic tumor cell death and decreases regulatory T cell (Treg) numbers in mice with transplantable tumors. Using the ret transgenic murine melanoma model that resembles human melanoma, we detected no beneficial antitumor effects with such treatment, despite a decrease in Tregs. On the contrary, low-dose CP enhanced the production of chronic inflammatory mediators in melanoma lesions associated with increased accumulation of Gr1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs), which exhibit elevated suppressive activity and nitric oxide (NO) production as well as inhibition of T-cell proliferation. Moreover, the frequencies of CD8(+) T cells in the tumors and their ability to produce perforin were decreased. To study whether the observed CP-induced MDSC expansion and activation also occurs under chronic inflammatory tumor-free conditions, mice exhibiting chronic inflammation were treated with CP. Similar to tumor-bearing mice, CP-treated inflamed mice displayed elevated levels of MDSCs with enhanced production of NO, reactive oxygen species, and a suppressed in vivo natural killer (NK) cell cytotoxic activity indicating CP effects on the host immune system independent of the tumor. We suggest that melanoma therapy with low-dose CP could be efficient only when combined with the neutralization of MDSC immunosuppressive function and chronic inflammatory microenvironment.

Application of paclitaxel in low non-cytotoxic doses supports vaccination with melanoma antigens in normal mice.

Chemotherapeutic agents such as paclitaxel applied in ultra-low, non-cytotoxic doses were previously shown to stimulate dendritic cell activity and anti-tumor immune responses upon vaccination in mouse transplantable tumor models. However, the mechanisms of these alterations-termed chemoimmunomodulation or chemomodulation-are still not clear. This study investigated the effect of paclitaxel applied in ultra-low, non-cytotoxic doses on the efficiency of immunization of healthy C57BL/6 mice with the peptide derived from tyrosinase related protein (TRP)-2 as a model melanoma antigen. Using an IFNγ ELISPOT assay, it was found that administration of 1 mg paclitaxel/kg in combination with the peptide vaccination strongly increased the frequencies of TRP-2 specific spleen T-cells as compared to levels due to the vaccination alone. This was associated with a significant decrease in the levels of regulatory T-cells (T(reg)) and immature myeloid cells (known as a counterpart of myeloid derived suppressor cells [MDSC] in healthy mice). Such impairments of potential immunosuppressive cells were found to correlate with a strong increase in the amount of effector CD8+ and CD4+ T-cells in the bone marrow and spleen. Furthermore, in paclitaxel-treated mice, a significant augmentation of natural killer (NK) cell numbers in the bone marrow and their ability to produce IFNγ were observed. In addition, the level of NK-T-cells in the lymph nodes was also increased. It is suggested that paclitaxel applied in ultra-low, non-cytotoxic doses may potentially enhance the efficacy of anti-tumor vaccinations by neutralizing immunosuppressive T(reg) and MDSC populations in tumor-bearing hosts.

The CD6 scavenger receptor is differentially expressed on a CD56 natural killer cell subpopulation and contributes to natural killer-derived cytokine and chemokine secretion.

The CD6 scavenger receptor is known to be expressed on virtually all T cells and is supposed to be involved in costimulation, synapse formation, thymic selection and leukocyte migration. Here, we demonstrate that CD6 is differentially expressed by a subpopulation of peripheral CD56(dim) natural killer (NK) cells and absent on CD56(bright) NK cells. CD56(dim)CD16(+) cells represent the major NK subset in the periphery, and most cells within this group are positive for CD6. Most killer immunoglobulin-like receptor- and immunoglobulin-like transcript-positive cells also belong to the CD6(+) subpopulation, as expected from their restricted expression on CD56(dim) NK cells. In addition, CD6(+) NK cells are similar to the newly identified CD94(low)CD56(dim) NK subpopulation and most distant from the recently defined CD27(+) NK subpopulation based on the reverse correlation of expression between CD6 and CD27, a marker associated primarily with CD56(bright) NK cells. With respect to CD6 function on NK cells, direct CD6 triggering did not result in degranulation but induced secretion of cytokines (interferon-γ and tumor necrosis factor-α) and chemokines [CXCL10 (IP-10), CXCL1 (GRO-α)]. Thus, CD6 expression on peripheral NK cells marks a novel CD56(dim) subpopulation associated with distinct patterns of cytokine and chemokine secretion.