Immune Niches Research Articles

Implantable Synthetic Immune Niche for Spatiotemporal Modulation of Tumor-Derived Immunosuppression and Systemic Antitumor Immunity: Postoperative Immunotherapy.

The development of biomaterial-based immune niches that can modulate immunosuppressive factors in tumor microenvironment (TME) will be a key technology for improving current cancer immunotherapy. Here, implantable, engineered 3D porous scaffolds are designed to generate synergistic action between myeloid-derived suppressor cell (MDSC)-depleting agents, which can accommodate the establishment of a permissive immunogenic microenvironment to counteract tumor-induced immunosuppression, and cancer vaccines consisting of whole tumor lysates and nanogel-based adjuvants, which can generate tumor antigen-specific T cell responses. The local peritumoral implantation of the synthetic immune niche (termed immuneCare-DISC, iCD) as a postsurgical treatment in an advanced-stage primary 4T1 breast tumor model generates systemic antitumor immunity and prevents tumor recurrence at the surgical site as well as the migration of residual tumor cells into the lungs, resulting in 100% survival. These therapeutic outcomes are achieved through the inhibition of immunosuppressive MDSCs in tumors and spleens by releasing gemcitabine and recruitment/activation of dendritic cells, enhanced population of CD4+ and CD8+ T cells, and increased IFN-γ production by cancer vaccines from the iCD. This combined spatiotemporal modulation of tumor-derived immunosuppression and vaccine-induced immune stimulation through the iCD is expected to provide an immune niche for prevention of postoperative tumor recurrence and metastasis.

Spatial reconstruction of immune niches by combining photoactivatable reporters and scRNA-seq.

Cellular functions are strongly dependent on surrounding cells and environmental factors. Current technologies are limited in their ability to characterize the spatial location and gene programs of cells in poorly structured and dynamic niches. We developed a method, NICHE-seq, that combines photoactivatable fluorescent reporters, two-photon microscopy, and single-cell RNA sequencing (scRNA-seq) to infer the cellular and molecular composition of niches. We applied NICHE-seq to examine the high-order assembly of immune cell networks. NICHE-seq is highly reproducible in spatial tissue reconstruction, enabling identification of rare niche-specific immune subpopulations and gene programs, including natural killer cells within infected B cell follicles and distinct myeloid states in the spleen and tumor. This study establishes NICHE-seq as a broadly applicable method for elucidating high-order spatial organization of cell types and their molecular pathways.

Abstract A34: The identification of combinatorial therapeutic approaches with BCR inhibitors in B cell malignancies

Abstract Introduction: It was shown that BCR inhibitors such as ibrutinib interrupt microenvironmental interactions and mobilize malignant B cells of chronic lymphocytic leukemia (CLL) from immune niches into the blood stream. It has been suggested that this mobilization will sensitize malignant B cells to other drugs, providing a possibility to introduce synthetically lethal combinations of drugs with an acceptable toxicity profile. This might be limited to a subgroup of CLL patients with specific underlying molecular mechanisms of disease biology or reaction to BCR inhibitors. The aim of this study was to test for the effect of ibrutinib on the expression of genes and activity of pathways that could be potentially targeted by clinically available drugs to achieve highly potent combinations. Results: We performed gene expression profiling in samples obtained from CLL patients treated with ibrutinib as a single agent (pre-ibrutinib vs. day 15 and/or week 5/12/15; N=12 pairs) to identify potential therapeutic targets. We have primarily focused on analyzes of cell-surface proteins and anti-apoptotic Bcl2 family members, since therapeutic antibodies and small molecular inhibitors for these molecules are available. We have analyzed changes in >30 cell-surface molecules, 8 members of the Bcl2 family, and members of other potentially relevant pathways (chemokines, adhesion, Akt, Erk, Ras, NFkB). Notably, we have observed that the mRNA and B cell-surface levels of CD20 are strongly down-modulated with ibrutinib, which indicates that the previously suggested combination of ibrutinib and anti-CD20 antibodies (such as rituximab or ofatumumab) is likely not ideal. Indeed, we showed that CLL cells with lower levels of CD20 are more resistant to rituximab in vitro, but also in vivo (P<0.01). We also showed that the effect of ibrutinib on CD20 levels is mediated by inhibition of CXCR4/SDF-1 axis, which impairs the SDF-1α (ligand for CXCR4) mediated regulation of CD20 expression (P<0.01). Notably, it has been shown that addition of rituximab to ibrutinib in vivo is able to partially eliminate CLL cells despite lower levels of CD20 suggesting that there must be other mechanism of ibrutinib action that allows for the effect of rituximab. The screening of anti-apoptotic molecules revealed that Mcl1 levels were significantly down-modulated after ibrutinib treatment in vivo (P=0.002), and ibrutinib also inhibited Mcl1 induction in CLL cells co-cultured with stromal cells (HS-5). As Mcl1 was described to directly protect CLL cells from rituximab, we suggest that ibrutinib-mediated Mcl1 reduction is a mechanism that facilitates rituximab efficacy. On the other hand we have shown that several members of Bcl2 family are up-regulated in CLL samples obtained after >3 weeks on ibrutinib therapy. We have also shown that interactions of stromal cells (HS-5) with CLL cells lead to up-regulation of these molecules while ibrutinib inhibits this in vitro (P<0.01). This suggest that the observed in vivo up-regulation of specific Bcl2 family members is due to an adaptation of B cells or selection of malignant B cells with high-level expression of anti-apoptotic molecules sufficient to survive the BCR signalling inhibition. We observed that the survival of CLL cells treated with ibrutinib is dependent on Bcl2, and subsequent Bcl2 inhibition can act synergistically with ibrutinib in B cell lines and CLL samples. These anti-apoptotic molecules therefore represent primary candidates with synthetically lethal potential in combination with ibrutinib. Additionally, we have identified dependence of a subtype of CLL cases on integrin signalling. The inhibition of integrin VLA-4 with anti-VLA4 antibody or integrin-associated kinase overcame the resistance of CLL cells to chemotherapy drugs and also to anti-CD20 antibodies, and synergized with the effects of ibrutinib. Conclusion: We have performed gene expression analysis of primary samples obtained from patients on ibrutinib, and identified gene expression changes that are associated with adaptation of B cells and/or selection of B cell resistant to BCR inhibition. We have shown that malignant B cells are dependent on integrin-signalling and the up-regulation of Bcl2 protein, which makes them synergistically lethal targets. Supported by: The results of this research have been acquired within CEITEC 2020 (LQ1601) project with financial contribution made by the Ministry of Education, Youths and Sports of the Czech Republic within special support paid from the National Programme for Sustainability II funds. Citation Format: Gabriela Pavlasova, Katerina Musilova, Vaclav Seda, Katerina Cerna, Eva Vojackova, Veronika Svobodova, Marek Mraz. The identification of combinatorial therapeutic approaches with BCR inhibitors in B cell malignancies [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A34.

Synthetic immune niches for cancer immunotherapy.

Cancer immunotherapy can successfully promote long-term anticancer immune responses, although there is still only a limited number of patients who benefit from such treatment, and it can sometimes have severe treatment-associated adverse events. Compared with systemic immunomodulation, local immunomodulation may enable more effective treatment at lower doses and, at the same time, prevent systemic toxicity. Local delivery of engineered three-dimensional scaffolds may fulfil this role by acting as synthetic immune niches that boost anticancer immunity. In this Opinion article, we highlight the potential of scaffold-based adoptive cell transfer and scaffold-based cancer vaccines that, although applied locally, can promote systemic antitumour immunity. Furthermore, we discuss how scaffold-based cancer immunotherapy may contribute to the development of the next generation of cancer treatments.

Abstract 1479: Differential expression of microRNAs in transformation of follicular lymphoma to diffuse large B cell lymphoma

Abstract MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression, and are frequently aberrantly expressed in cancer. We aimed to understand their role in the transformation of indolent follicular lymphoma (FL) into an aggressive diffuse large B cell lymphoma. This happens in ~3% of cases per year during the course of the disease, and is associated with median survival of only 2 years. The NGS revealed number of aberrations associated with transformed FL (tFL), including frequent high-level activity of MYC (amplifications, translocations, and mutations) or loss of DNA damage regulators (p53, CDKN2A/B). Firstly, we performed a miRNA profiling (TaqMan miRNA Arrays) in paired FL and tFL samples (N=8 pairs). This revealed a relatively small group of 5 miRNAs that are consistently differentially expressed in tFL (P<0.05, fold-change >1.5). Since the most frequently acquired aberration in tFL is the high-level activity of MYC we performed a correlation analysis of MYC levels and expression of these miRNAs in additional samples of FL, tFL, and CLL samples with/without MYC duplication (N=40 FL/tFL, N=39 CLL). This revealed that at least one of these miRNAs is significantly down-modulated (P<0.05) in cases with high-levels of MYC. The MYC-mediated repression of miRNA levels was also observed (P<0.05) in B cells from transgenic MYC over-expressing mice (MYC controlled by an Ig-alpha enhancer) in comparison to wild-type animals (samples obtained from young animals before occurrence of any malignancy). We have further shown that the levels of this miRNA affect B cell proliferation in vitro, and its low-levels associate with percentage of Ki67 positive cells in FL samples (P<0.005). Moreover, low levels of tFL-associated miRNA were present in FL cases with a shorter overall survival (P<0.01), and its expression directly affected BCR signalling (calcium flux assay after anti-IgM). We have shown that the expression of this miRNA is not only down-modulated by high-level MYC expression, but also by B cell adhesion to stromal cells in co-culture in vitro (HS-5 stromal cells). This suggests that its normal physiological function might be related to regulation of B cell functions in the context of immune niches, and this might play a role in FL progression and transformation. It remains to be elucidated what other molecular mechanisms ensure low-level expression of the studied miRNA in cases that do not harbor MYC over-expression, and what pool of target mRNAs is regulated by this miRNA in FL cells.This work was supported by: the Ministry of Health of the Czech Republic, grant nr. 16-29622A. All rights reserved. contact: marek.mraz@email.cz Citation Format: Katerina Musilova, Gabriela Pavlasova, Vaclav Seda, Eva Vojackova, Katerina Cerna, Veronika Svobodova, Robert Pytlik, Vit Prochazka, Zuzana Prouzova, Sarka Pospisilova, Lenka Zlamalikova, Heidi Mocikova, Lenka Kruzova, Marie Jarosova, Andrew Evans, Clive Zent, Leos Kren, Marek Trneny, Jiri Mayer, Andrea Janikova, Marek Mraz. Differential expression of microRNAs in transformation of follicular lymphoma to diffuse large B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1479. doi:10.1158/1538-7445.AM2017-1479

Mechanism and Activity of ILC2 Cells Post Allo-BMT

The reconstitution of specific immune niches after bone marrow transplant is mediated by donor immune cells repopulating specific microenvironments. Here, we demonstrate that type 2 innate lymphoid cells (ILC2) in the GI tract are rapidly eliminated by irradiation and/or chemotherapy and are not repopulated in the first month post-transplant by donor bone marrow cells. In contrast, ILC2 cells in the lung are not sensitive to conditioning therapy and quickly recover quantitatively post-transplant. Donor ILC2 cell infusion with donor T cells and TCD bone marrow has been shown by us to markedly diminish lethality and clinical score associated with acute GvHD. However, the mechanism for that finding was unclear. Here, we demonstrate that donor ILC2 infusion enhances the persistence of GI tract, but not splenic or pulmonary MDSCs (Fig 1a). This persistence required the expression of IL-13 by the ILC2 cells (Fig 1b). Absence of IL-13 production by ILC2 cells greatly diminished the activity of those cells to prevent GvHD (Fig 1c). ILC2 cell infusion decreased the number of T cells generating IFN-γ and IL-17A in the GI tract. However, we found no difference in the number of IFN-γ or IL-17A generating T cells in the GI tract after the infusion of IL-13-/- ILC2 cells.To evaluate the function of ILC2 cells in MDSC biology we performed in vitro quantitation of MDSCs in co-culture with ILC2 cells. MDSCs cultured with WT ILC2s, survived significantly better than MDSCs cultured alone, MDSCs cultured with IL-13 and MDSC cultured in the presence of IL-7 and IL-33. In addition, MDSCs co-cultured with WT ILC2s survived significantly better than those cultured with IL-13-/- ILC2s. Surprisingly, enhancement of MDSC survival requires cell-to-cell contact with ILC2s, as seen when either WT or IL-13-/- ILC2s and MDSCs are cultured in plates across semi-permeable membranes.ILC2s have been shown to have a role in intestinal barrier repair via amphiregulin (Areg). When compared to untreated recipients, ILC2 treated recipients showed significantly enhance barrier function which can be observed as early as 12 days post-transplant and as late as day 20. Interestingly, recipients of Areg deficient ILC2s show improved survival compared to control treatment but diminished survival compared to recipients of WT ILC2 cells.Several cell types have been shown to suppress aGvHD, including MDSCs and regulatory T cells (Treg). To evaluate the function of ILC2 cells as treatment, we compared them to MDSC and Treg infusion. When used to treat aGvHD on day 7 post-transplant, IL-13 derived MDSCs have no effect on recipient survival. In contrast ILC2 infusion results in a significant prolongation of survival compared to MDSC infusion with approximately 35% of recipient mice surviving past day 50. When compared to Treg infusion (1:1 ratio with Tcons) both ILC2 and Treg infusions block aGvHD. However, unlike ILC2 infusion in which no mice develop P815 tumors post-transplant, 80% of mice given Tregs developed tumors. At a lower Treg dose (1:4) the GvL response was restored but without the beneficial effect on GvHD.In summary, ILC2s represent a novel cell population that are required for recovery of GI tract homeostasis following allo-SCT. ILC2s play a pivotal role in the development of donor MDSCs and intestinal barrier repair. Additional investigation should be focused on the recovery of specific immunosuppressive cell populations from donor bone marrow cells after allo-SCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 3291: The expression of CD20 on malignant B cells is regulated by chemokine signaling through the CXCR4/SDF-1 axis: implications for targeting the microenvironmental interactions

Abstract The introduction of anti-CD20 antibodies has significantly improved the outcome of patients with chronic lymphocytic leukemia (CLL) and B-cell non-Hodgkin lymphomas. The aim of this study was to analyze molecular pathways that influence expression of CD20 since this is largely unknown. This is of a great clinical interest, as combinatorial therapy of novel BCR-signaling inhibitors ibrutinib and idelalisib currently focuses mainly on the use with anti-CD20 antibodies. Firstly, we analyzed samples obtained from CLL patients treated with ibrutinib and showed that administration of ibrutinib in vivo leads to CD20 down-modulation (P<0.0001). That implies that CD20 expression might be regulated by a yet unknown mechanism in the context of immune niches. Therefore, we focused on determining the effect of microenvironmental interactions on CD20 expression on malignant B cells. We co-cultured primary CLL cells with the bone marrow stromal cell line HS-5. This revealed that CLL cells co-cultured with HS-5 had higher surface CD20 expression compared to control cells cultured on plastic (P<0.01). Moreover, CLL cells that have been recently released from the lymph node microenvironment to the peripheral blood (CXCR4dimCD5bright) had ∼2-fold higher surface CD20 expression when compared to CLL cells circulating in blood of the same patient for a longer time (CXCR4brightCD5dim cells; P<0.0001). Further, sorted CXCR4dimCD5bright CLL cells had also ∼2 times higher CD20 mRNA expression (P = 0.002) suggesting that changes in CD20 expression were rather due to changes in its mRNA levels than surface modulation. As CD20 expression was decreasing with the transition from CXCR4dim to CXCR4bright CLL cells, we hypothesized that the CXCR4/SDF-1 axis is directly implicated in CD20 regulation. Indeed, CLL cells treated with SDF-1α (CXCL12), a ligand for CXCR4 produced by stromal cells, significantly up-regulated surface CD20 (P<0.05). On the contrary, the treatment with plerixafor, CXCR4 antagonist, inhibited this SDF-1α mediated up-regulation of CD20 which proves that SDF-1α binding to CXCR4 is directly involved in CD20 regulation. Altogether, the CD20 levels are up-regulated within the context of microenvironmental interactions through the CXCR4/SDF-1 axis, and the impairment of microenvironmental interactions mediated by ibrutinib down-regulates CD20 expression. This study reveals a novel regulation of CD20 expression in the context of immune niches, which has important implications for CD20-targeting antibodies and the use of BCR-inhibitors in combination. Supported by: SoMoPro II-no.4SGA8684; NGS-PTL(306242); EHA Fellowship award; Ministry of Health of CR (16-29622A); Academy of Sciences of CR (16-13334Y); Ministry of Education, Youth and Sports, grant LD15144 (COST CZ); MUNI/A/1028/2015; CZ.1.05/1.1.00/02.0068; G.P. is supported by Ostrava city. Citation Format: Gabriela Pavlasova, Marek Borsky, Vaclav Seda, Katerina Cerna, Jitka Osickova, Michael Doubek, Yvona Brychtova, Jiri Mayer, Sarka Pospisilova, Matthew S. Davids, Jennifer R. Brown, Marek Mraz. The expression of CD20 on malignant B cells is regulated by chemokine signaling through the CXCR4/SDF-1 axis: implications for targeting the microenvironmental interactions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3291.

Papers of note in Science

This week’s articles highlight immune cell niches and microbe-induced mucus secretion in the intestine and the structural basis of kinase activation by chaperones.

Introduction to Clinical Immunology: Overview of the Immune Response, Autoimmune Conditions, and Immunosuppressive Therapeutics for Rheumatic Diseases

The immune system is a complex network of cells and mediators that must balance the task of protecting the host from invasive threats. From a clinical perspective, many diseases and conditions have an obvious link to improper functioning of the immune system, and insufficient immune responses can lead to uncontrolled acute and chronic infections. The immune system may also be important in tumor surveillance and control, cardiovascular disease, health complications related to obesity, neuromuscular diseases, depression, and dementia. Thus, a working knowledge of the role of immunity in disease processes is becoming increasingly important in almost all aspects of clinical practice. This review provides an overview of the immune response and discusses immune cell populations and major branches of immunity, compartmentalization and specialized immune niches, antigen recognition in innate and adaptive immunity, immune tolerance toward self antigens, inflammation and innate immune responses, adaptive immune responses and helper T (Th) cell subsets, components of the immune response that are important targets of treatment in autoimmune diseases, mechanisms of action of biologics used to treat autoimmune diseases and their approved uses, and mechanisms of other drugs commonly used in the treatment of autoimmune diseases. Figures show the development of erythrocytes, platelets, lymphocytes, and other immune system cells originating from hematopoietic stem cells that first reside in the fetal liver and later migrate to the bone marrow, antigen–major histocompatibility complex recognition by T cell receptor control of T cell survival and activation, and Th cells as central determinants of the adaptive immune response toward different stimuli. Tables list cell populations involved in innate and adaptive immunity, pattern recognition receptors with known ligands, autoantibody-mediated human diseases: examples of pathogenic mechanisms, selected Food and Drug Administration–approved autoimmune disease indications for biologics, and mechanism of action of biologics used to treat autoimmune diseases. This review contains 3 highly rendered figures, 5 tables, and 64 references.

Introduction to Clinical Immunology: Overview of the Immune Response, Autoimmune Conditions, and Immunosuppressive Therapeutics for Rheumatic Diseases

The immune system is a complex network of cells and mediators that must balance the task of protecting the host from invasive threats. From a clinical perspective, many diseases and conditions have an obvious link to improper functioning of the immune system, and insufficient immune responses can lead to uncontrolled acute and chronic infections. The immune system may also be important in tumor surveillance and control, cardiovascular disease, health complications related to obesity, neuromuscular diseases, depression, and dementia. Thus, a working knowledge of the role of immunity in disease processes is becoming increasingly important in almost all aspects of clinical practice. This review provides an overview of the immune response and discusses immune cell populations and major branches of immunity, compartmentalization and specialized immune niches, antigen recognition in innate and adaptive immunity, immune tolerance toward self antigens, inflammation and innate immune responses, adaptive immune responses and helper T (Th) cell subsets, components of the immune response that are important targets of treatment in autoimmune diseases, mechanisms of action of biologics used to treat autoimmune diseases and their approved uses, and mechanisms of other drugs commonly used in the treatment of autoimmune diseases. Figures show the development of erythrocytes, platelets, lymphocytes, and other immune system cells originating from hematopoietic stem cells that first reside in the fetal liver and later migrate to the bone marrow, antigen–major histocompatibility complex recognition by T cell receptor control of T cell survival and activation, and Th cells as central determinants of the adaptive immune response toward different stimuli. Tables list cell populations involved in innate and adaptive immunity, pattern recognition receptors with known ligands, autoantibody-mediated human diseases: examples of pathogenic mechanisms, selected Food and Drug Administration–approved autoimmune disease indications for biologics, and mechanism of action of biologics used to treat autoimmune diseases. This review contains 3 highly rendered figures, 5 tables, and 64 references.

Glioma Stem Cells and Their Microenvironments: Providers of Challenging Therapeutic Targets.

Malignant gliomas are aggressive brain tumors with limited therapeutic options, possibly because of highly tumorigenic subpopulations of glioma stem cells. These cells require specific microenvironments to maintain their “stemness,” described as perivascular and hypoxic niches. Each of those niches induces particular signatures in glioma stem cells (e.g., activation of Notch signaling, secretion of VEGF, bFGF, SDF1 for the vascular niche, activation of HIF2α, and metabolic reprogramming for hypoxic niche). Recently, accumulated knowledge on tumor-associated macrophages, possibly delineating a third niche, has underlined the role of immune cells in glioma progression, via specific chemoattractant factors and cytokines, such as macrophage-colony stimulation factor (M-CSF). The local or myeloid origin of this new component of glioma stem cells niche is yet to be determined. Such niches are being increasingly recognized as key regulators involved in multiple stages of disease progression, therapy resistance, immune-escaping, and distant metastasis, thereby substantially impacting the future development of frontline interventions in clinical oncology. This review focuses on the microenvironment impact on the glioma stem cell biology, emphasizing GSCs cross talk with hypoxic, perivascular, and immune niches and their potential use as targeted therapy.

Microenvironmental Interactions up-Regulate CD20 Expression in CLL B Cells through the CXCR4/SDF-1 Axis: Implications for CD20-Targeting Antibodies and the Use of BCR-Inhibitors in Combination

Abstract The aim of this study was to investigate the role of microenvironmental interactions in the regulation of CD20 expression, since down-modulation of CD20 in the context of immune niches would be a straightforward explanation for the stroma-mediated rituximab resistance described by us and others (Mraz et al., BJH, 2011; Buchner et al., BJH, 2010; Marquez et al., BJH, 2015). Firstly, we co-cultured primary CLL cells with the bone marrow stromal cell line HS-5 for 24/48/72 hrs and analyzed CD20 surface expression by flow cytometry. Suprisingly, CD20 levels were significantly up-regulated (P=0.03) on CLL cells co-cultured with HS-5 in comparison to CLL cells cultured on plastic. The changes in CD20 expression levels are likely to play a direct role in microenvironmental interactions and especially BCR signaling in immune niches (Uchida et al., Int Immunol, 2004). To test this hypothesis we assessed the CD20 expression on CLL cell populations defined according to CXCR4 and CD5 levels. The low-level surface expression of chemokine receptor CXCR4 and high-level expression of activation molecule CD5 can characterize B cells that have recently exited the lymph nodes (Calissano et al., Mol Med, 2011). CXCR4dim CD5bright cells had ~2-fold higher surface CD20 expression when compared to CXCR4bright CD5dim CLL cells circulating in the blood of the same patient for a longer time (P<0.0001, N=21) and CD20 expression clearly decreased with the transition of CLL cells from CXCR4dim to CXCR4bright together with the decrease of CD5 expression. Further, sorted CXCR4dim CD5bright CLL cells had also ~2 times higher CD20 mRNA expression (P=0.002, N=8) suggesting that changes in CD20 expression were due to changes in its mRNA levels rather than a surface modulation. This led us to hypothesize that CXCR4/SDF-1 is directly implicated in CD20 regulation. Indeed, CLL cells treated with SDF-1α (CXCL12), a ligand for CXCR4 which is produced by HS-5, up-regulated surface CD20 (P=0.03). We also observed that CLL cells with higher expression of CD20 had higher levels of surface Ig (P=0.0015, N=12) and this was coupled with higher responsiveness to BCR crosslinking with anti-IgM as measured by calcium flux (P=0.005). Therefore, we investigated the effect of ibrutinib on CD20 levels as ibrutinib is a BCR-signaling pathway inhibitor and prevents CLL cells from responding to microenvironmental stimuli, including chemokine signaling. We observed that treatment of CLL cells with ibrutinib in vitro resulted in CD20 down-modulation (P<0.001). The levels of CD20 in CLL cells were also strongly down-modulated in samples obtained before vs. during ibrutinib treatment of CLL patients in vivo (pre- ibrutinib vs. day 15 and week 5/12, N=8; P=0.02, P=0.01, P=0.07, respectively). These data suggest that a reduction of the target antigen (CD20) is not the cause for rituximab resistance in the context of immune niches, and the sustained activity of ibrutinib and rituximab combinations in clinical trials (Burger et al., Lancet Oncol, 2014) suggests that ibrutinib has other mechanisms that allow for rituximab efficacy. We focused on the regulation of anti-apoptotic molecules, namely Mcl1, since that was shown to protect from rituximab-induced apoptosis and to be rapidly down-modulated by rituximab infusion in vivo (Byrd et al., Blood, 2002; Hussain et al., Clin Cancer Res, 2007). Indeed, we observed higher levels of Mcl1 mRNA in the CXCR4dim CD5bright CLL subpopulation (N=8, P=0.04), and in CLL cells co-cultured with stromal cells. Importantly, Mcl1 was down-regulated after ibrutinib treatment both in vitro and in ibrutinib treated patients. Conclusion: Microenvironmental interactions up-regulate CD20 expression in CLL B cells through the CXCR4/SDF-1 axis, and the ibrutinib-mediated impairment of microenvironmental interactions down-regulates CD20 expression. This study reveals a novel regulation of CD20 levels in the context of immune niches, which has important implications for CD20-targeting antibodies and the use of BCR-inhibitors in combination. Supported by: SoMoPro II-no.4SGA8684 (cofinanced by the European Union and the South-Moravian Region); NGS-PTL(306242); EHA Fellowship award; IGA MZ CR NT11218-6/2010; MUNI/A/1180/2014; CZ.1.05/1.1.00/02.0068; G.P. is a city of Ostrava scholarship holder. #G.P. and V.S. contributed equally to this study. contact: marek.mraz@email.cz Disclosures Mayer: Janssen: Research Funding. Davids:Genentech: Other: ad board; Pharmacyclics: Consultancy; Janssen: Consultancy.

To serve and to protect: the role of decidual innate immune cells on human pregnancy.

The maternal-fetal interface undergoes dynamic changes that promote successful development of the embryo/fetal allograft during pregnancy. This immune privilege of the conceptus is mediated through local and systemic cellular responses. In species in which endometrial decidualization accompanies pregnancy, unique immune cell niches are found. Many studies have addressed the enigmatic roles of uterine (u)NK cells as killers and helpers because they are frequently found in the uterine lining and decidua of normal and pathological pregnancies. Accumulating evidence indicates that uNK cells are induced and transformed by sensing signals within their microenvironment to both protect the mother from the fetal allograft and support the fetus during its development. Here, we review the mechanisms that modulate these functions of uNK cells during pregnancy. We suggest that uNK cells must be tightly regulated in order to serve these two roles and support a healthy pregnancy.

MicroRNAs in B-cell lymphomas: how a complex biology gets more complex.

MicroRNAs (miRNAs) represent important regulators of gene expression besides transcriptional control. miRNA regulation can be involved in the cell developmental fate decisions, but can also have more subtle roles in buffering stochastic fluctuations in gene expression. They participate in pathways fundamental to B-cell development like B-cell receptor (BCR) signalling, B-cell migration/adhesion, cell-cell interactions in immune niches, and the production and class-switching of immunoglobulins. miRNAs influence B-cell maturation, generation of pre-, marginal zone, follicular, B1, plasma and memory B cells. In this review, we discuss miRNAs with essential functions in malignant B-cell development (such as miR-150, miR-155, miR-21, miR-34a, miR-17-92 and miR-15-16). We also put these miRNAs in the context of normal B-cell differentiation, as this is intimately connected to neoplastic B-cell development. We review miRNAs' role in the most common B-cell malignancies, including chronic lymphocytic leukaemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL). We focus on miR-contribution to the regulation of important signalling pathways (such as NF-κB, PI3K/AKT and TGF-β), BCR signalling and its modulators (such as PTEN, SHIP-1, ZAP-70, GAB1 and BTK), anti- and pro-apoptotic proteins (such as BCL2, MCL1, TCL1, BIM, p53 and SIRT1) and transcription factors (such as MYC, MYB, PU.1, FOXP1 and BCL6). We also discuss the association of miRNAs' expression levels with the patients' survival and response to therapy, summarizing their potential use as predictive and prognostic markers. Importantly, the targeting of miRNAs (like use of anti-miR-155 or miR-34a mimic) could provide a novel therapeutic approach as evidenced by tumour regression in xenograft mouse models and initial promising data from clinical trials.

Su1266 In Vitro Generated Regulatory T Cells From Blood Suppress Mucosal Effector T Cells in Crohn's Disease Patients

Background: Thymically derived CD4+CD25hiCD127loFOXP3+ regulatory T cells (Tregs) maintain immunological tolerance and prevent inappropriate activation of multiple immune cell types. Quantitative and qualitative Treg defects are seen in inflamed Crohn's (CD) mucosa, suggesting that insufficient Treg-mediated suppressor function contributes to chronic mucosal inflammation in CD. Consequently, cell-based therapy with autologous Tregs to restore mucosal Treg numbers and function is conceptually attractive. The recent use of in vitro expanded umbilical cord blood (UCB) Tregs to prevent GvHD following UCB transplantation confirmed the safety of this approach in humans. The optimal method of generating Tregs in vitro from CD patients to produce a homogenous, phenotypically stable yet functionally suppressive population on expansion is unknown, as is the effect of these cells on target organs: mesenteric lymph node (MLN) and lamina propria (LP) immune cells in inflamed CD gut. Aim: To define the optimal method of GMP-compatible Treg isolation and expansion from CD patients' blood with reference to homogeneity, phenotypic stability and suppressive ability of the cell product. Results: Tregs were expanded for 2028 days in X-VIVO15 with 5% human AB serum, IL-2 and Rapamycin. Tregs initially isolated using GMP-compatible MACS-based enrichment had unacceptably high CD8+ contamination (.20%) and high expression of IL-17 and IFNγ (n=5 independent experiments). CD4+CD25+CD127lo Tregs were then sorted from CD blood on the basis of CD45RA expression and expanded a median of 127-fold (IQR 58-531) to yield a .97% CD4+CD25hiCD127loFOXP3+ population (n=10). Expanded CD45RA+ Tregs expressed functionally relevant markers including CTLA-4, ICOS, CD27 and CD62L ( .97% each) and TGF-β but not IL-10. CD45RA+ Tregs expressed ,1% IL-17 by FACS and had greater phenotypic stability than expanded CD45RATregs, with less spontaneous (p,0.05) and induced (p,0.01) IL-17 production by ELISA. CD45RA+ Tregs highly suppressed autologous CD4+CD25proliferation at 96h (median=97.3% [96.6%-99.1%] at 1:1 ratio); and were more effective than CD45RATregs at suppressing CD4+CD25activation (p=0.003), assessed by CD154 expression at 7h. Importantly, using CD3+ T responders from inflamed Crohn's resections, CD45RA+ Tregs suppressed MLN CD3+ proliferation (88.4% [53.8-93.2%]), MLN CD154 expression (82.9% [60.1-87.6%]) and LP CD3+ CD154 expression (64.3% [64.377 . 9%] ) i n a do s e d ep enden t manne r . Conc lu s i ons : FACS so r t i n g o f a CD4+CD25hiCD127loCD45RA+ precursor population is required to produce a homogenous, phenotypically stable in vitro expanded cell product from CD patients. For the first time, we show that in vitro expanded Tregs suppress immune cell activation in relevant mucosal immune niches, strengthening the rationale for clinical trials of autologous in vitro expanded Tregs in CD.

Abstract 3531: Changes in immune cell populations in relapsed/refractory CLL patients treated with a Bruton's Tyrosine Kinase (BTK) Inhibitor, Ibrutinib (PCI-32765), in combination with Bendamustine and Rituximab (BR).

Abstract Although chemoimmunotherapy has led to higher response rates in chronic lymphocytic leukemia (CLL), most patients relapse due to emergence of resistant subclones. Here, we analyzed changes in immune cell (IC) populations of 29 subjects with relapsed/refractory CLL enrolled in PCYC-1108, a Phase 1b, open-label, parallel-group, nonrandomized, multicenter study of ibrutinib (PCI-32765) in combination with BR (O'Brien et al; ASCO 2012). Mononuclear cells were collected and cryopreserved at 4 time points: 1: just prior to therapy, 2: cycle 1 day 15 (d15), 3: at start of cycle 3 (d 60) and; 4: at the end of study (d 180). Based on a decrease in absolute lymphocyte count (ALC), 27/29 patients responded to this combination therapy. We analyzed residual B, T and monocyte populations and prognostic markers, CD38 and CD49d in each patient and grouped the cases based on percent reduction of CD19+CD5+ (“CLL B cells”) at all time points while on therapy. 10/29 cases (∼34%) showed > 50% reduction (Grp1) whereas 41% (12/29) showed greater retention of CLL cells (Grp2), and 7/29 cases (∼24%) showed variable changes (Grp 3). Median reduction in ALC in Grp1 was 97.8% (range: 68.3-99.75) vs 87.7% (range: 77.3- 99.6) for Grp2. Grp 1 cases showed a decrease in CD23+ and CD38+ CLL B cells at d15; although CD23+ cells decreased further CD38+ cells increased in 8/10 Grp1 cases. CD49d+ CLL cells remained stable in 6/10 Grp 1 and 11/12 Grp 2 cases but increased in 4/10 Grp1 cases. A marked increase in CD3+ and CD14+ cells accompanied the decrease in CLL B cells in cases in Grp1. Grp2 followed a different trend: CD3+ cells decreased until d60 and eventually increased, albeit mildly whereas their percentages of CD14+ cells did not change throughout therapy. Such dynamic changes in IC populations prompted us to assess the presence of dividing CLL cells, in the face of reduced ALC. Surprisingly, although Grp1 showed decreased percentages of CLL cells, 3/10 cases developed increased numbers of Ki-67+ CLL cells, achieving 19-43% at d180. In Grp2, only 0.1-4.0% Ki-67+ CLL B cells were found. BCR-induced phosphorylation of BTK was analyzed by phosphoflow. Both Grp1 and Grp2 exhibited increased BTK phosphorylation at d15 which decreased precipitously at subsequent time points. Together, these studies indicate that combining ibrutinib with BR effectively reduced numbers of circulating CLL cells in >93% of cases, but relative proportions of CLL cells only in ∼1/3 relapse/refractory CLL cases. Of note, the proportion of circulating CD3+, CD14+, and Ki-67+ CLL B cells, increased considerably in Grp 1 cases, possibly representing compensatory proliferative changes due to increased availability of immune niches and/or release of cells from niches. A post-protocol correlation of clinical parameters with changes in circulating immune cell populations is pending. Citation Format: Rajendra N. Damle, Sonal Temburni, Prachi Aggarwal, Susan O'Brien, Jacqueline Barrientos, Jennifer R. Brown, Ian W. Flinn, Paul Barr, Jan Burger, Jonathan W. Friedberg, Kanti R. Rai, Betty Chang, Danelle James, Joseph Buggy, Nicholas Chiorazzi. Changes in immune cell populations in relapsed/refractory CLL patients treated with a Bruton's Tyrosine Kinase (BTK) Inhibitor, Ibrutinib (PCI-32765), in combination with Bendamustine and Rituximab (BR). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3531. doi:10.1158/1538-7445.AM2013-3531

Targeting glioma stem cells: A novel framework for brain tumors

The past decade has seen a dramatic increase in stem cell research that focuses on glioma stem cells (GSC) and their mechanisms of action, revealing multiple potential targets for primary malignant brain tumors. Herein, we present a novel framework for considering GSC targets based on direct and indirect strategies. Direct strategies target GSC molecular pathways to overcome their resistance to radiation and chemotherapy, block their function or induce their differentiation. Indirect strategies target the microenvironment of the GSC, namely the perivascular, hypoxic and immune niches. Progress made on GSC targets is reviewed in detail and specific pathways are identified in context of the proposed framework. The potential barriers for translation to the clinical setting are also discussed. Overall, targeting GSC provides an unprecedented opportunity for revolutionary approaches to treat high-grade gliomas that continue to have a poor patient prognosis.

Perivascular clusters of dendritic cells provide critical survival signals to B cells in bone marrow niches

Beyond its established function in hematopoiesis, the bone marrow hosts mature lymphocytes and acts as a secondary lymphoid organ in the initiation of T cell and B cell responses. Here we report the characterization of bone marrow-resident dendritic cells (bmDCs). Multiphoton imaging showed that bmDCs were organized into perivascular clusters that enveloped blood vessels and were seeded with mature B lymphocytes and T lymphocytes. Conditional ablation of bmDCs in these bone marrow immune niches led to the specific loss of mature B cells, a phenotype that could be reversed by overexpression of the antiapoptotic factor Bcl-2 in B cells. The presence of bmDCs promoted the survival of recirculating B cells in the bone marrow through the production of macrophage migration-inhibitory factor. Thus, bmDCs are critical for the maintenance of recirculating B cells in the bone marrow.