Accessory Cell Types Research Articles

Autologous CD19-Targeted Chimeric Antigen Receptor (CAR)T-Cells as the Future of Systemic Lupus Erythematosus Treatment.

Systemic lupus erythematosus (SLE) is a chronic autoimmune illness with an unclear etiology and a range of clinical manifestations. The therapeutic results of current conventional treatments are frequently unsatisfactory. Many B-cell-directed immunotherapies have recently been discovered, as B cells play a key role in the pathogenesis of SLE. However, large-scale rituximab trials found that the antibody against CD20 was no better than a placebo. Autologous CAR T-cell therapy has garnered considerable interest and is considered a potential treatment option for SLE. CD19+CD20- B cells are thought to play an essential role in the onset and progression of SLE. CD19-targeted CAR T-cells destroy B cells without requiring an accessory cell type, thereby decreasing B cells more efficiently. Preclinical trials of CAR T-cells in mice have shown promising results against SLE. The review aimed to shed light on autologous CD19-targeted CAR T-cells as a potential treatment for SLE.

Chimeric antigen receptor T cell therapy for the treatment of systemic rheumatic diseases: a comprehensive review of recent literature.

Systemic rheumatoid diseases (SRDs) are autoimmune and inflammatory disorders that affect multiple organ systems, impacting patients' quality of life, and survival rates. Standard treatment requires continuous drug therapy and immunosuppression. Chimeric antigen receptor (CAR) T cell therapy has the potential to target and eliminate pathologically activated immune cells and re-establish tolerance in organs affected by dysregulated immunity, making them a promising treatment option for autoimmune diseases. In autoimmune diseases, CAR T cells have the advantage of being able to kill B cells effectively without the need for an accessory cell type. Additionally, CAR T cells targeting CD19 have shown promise in comprehensive B cell aplasia, preserving pre-existing humoral immunity, and specifically eliminating pathogenic B cells. CAR T cell therapy's limited use in SRDs is due to its inability to effectively target the various autoreactive lymphocytes present. Researchers are developing a universal CAR T cell therapy that detects and targets autoreactive lymphocytes using major epitope peptides, though further studies are required. Moreover, adoptive transfer of CAR-Tregs has shown promise for effectively reducing inflammation and treating autoimmunity. Through this exploration, the authors hope to provide a comprehensive understanding of the current state of research on this topic, identify areas for further study, and promote the advancement of CAR T cell therapy as a treatment option for SRDs.

Conserved transcriptional connectivity of regulatory T cells in the tumor microenvironment informs new combination cancer therapy strategies

While regulatory T (Treg) cells are traditionally viewed as professional suppressors of antigen presenting cells and effector T cells in both autoimmunity and cancer, recent findings of distinct Treg cell functions in tissue maintenance suggest that their regulatory purview extends to a wider range of cells and is broader than previously assumed. To elucidate tumoral Treg cell ‘connectivity’ to diverse tumor-supporting accessory cell types, we explored immediate early changes in their single-cell transcriptomes upon punctual Treg cell depletion in experimental lung cancer and injury-induced inflammation. Before any notable T cell activation and inflammation, fibroblasts, endothelial and myeloid cells exhibited pronounced changes in their gene expression in both cancer and injury settings. Factor analysis revealed shared Treg cell-dependent gene programs, foremost, prominent upregulation of VEGF and CCR2 signaling-related genes upon Treg cell deprivation in either setting, as well as in Treg cell-poor versus Treg cell-rich human lung adenocarcinomas. Accordingly, punctual Treg cell depletion combined with short-term VEGF blockade showed markedly improved control of PD-1 blockade-resistant lung adenocarcinoma progression in mice compared to the corresponding monotherapies, highlighting a promising factor-based querying approach to elucidating new rational combination treatments of solid organ cancers.

Non-cell-autonomous small RNA silencing in Arabidopsis female gametes

In recent years, small RNA movement has been both hypothesized and shown to be an integral part of the epigenetic DNA methylation reprogramming occurring during plant reproduction.1It was suggested that the release of epigenetic silencing in accessory cell types or tissues is necessary to reinforce epigenetic silencing in the gametes (egg cell and sperm cells), which would in turn ensure the genomic stability of the next generation plant.2,3 In Arabidopsis thaliana, small RNA (sRNA) movement was indeed shown to occur during male gametogenesis.4,5,6 However, the situation within the female gametophyte and in early seed development is mostly unknown. Here, we show that small RNAs can induce non-cell-autonomous silencing from the central cell toward the egg cell but also from the synergids to the egg cell and central cell. Our data show that in addition to the movement of sRNAs during pollen development, hairpin RNAs can have non-cell-autonomous effects in the female gametes.

Structural and Functional Thymic Biomarkers Are Involved in the Pathogenesis of Thymic Epithelial Tumors: An Overview

The normal human thymus originates from the third branchial cleft as two paired anlages that descend into the thorax and fuse on the midline of the anterior–superior mediastinum. Alongside the epithelial and lymphoid components, different types of lymphoid accessory cells, stromal mesenchymal and endothelial cells migrate to, or develop in, the thymus. After reaching maximum development during early postnatal life, the human thymus decreases in size and lymphocyte output drops with age. However, thymic immunological functions persist, although they deteriorate progressively. Several major techniques were fundamental to increasing the knowledge of thymic development and function during embryogenesis, postnatal and adult life; these include immunohistochemistry, immunofluorescence, flow cytometry, in vitro colony assays, transplantation in mice models, fetal organ cultures (FTOC), re-aggregated thymic organ cultures (RTOC), and whole-organ thymic scaffolds. The thymic morphological and functional characterization, first performed in the mouse, was then extended to humans. The purpose of this overview is to provide a report on selected structural and functional biomarkers of thymic epithelial cells (TEC) involved in thymus development and lymphoid cell maturation, and on the historical aspects of their characterization, with particular attention being paid to biomarkers also involved in Thymic Epithelial Tumor (TET) pathogenesis. Moreover, a short overview of targeted therapies in TET, based on currently available experimental and clinical data and on potential future advances will be proposed.

Osteoblast-Derived Paracrine and Juxtacrine Signals Protect Disseminated Breast Cancer Cells from Stress.

Simple SummaryBone metastasis is a debilitating, incurable complication that occurs in breast cancer patients with a variable period of latency after treatment of the primary tumor. The seeding of individual cancer cells in the bone marrow from the primary tumor occurs early in disease progression. How disseminated cancer cells are able to remain dormant in the bone marrow and then reactivate decades later to form destructive bone lesions is not fully understood. We have used model systems to identify bone resident cell types, and molecular mediators, that influence the survival and fate of disseminated breast cancer cells. The identification of such cellular and molecular support mechanisms can inform the design of new therapeutic strategies aimed at eliminating indolent cancer cells from the bone marrow, preventing progression to overt metastases.Metastatic breast cancer in bone is incurable and there is an urgent need to develop new therapeutic approaches to improve survival. Key to this is understanding the mechanisms governing cancer cell survival and growth in bone, which involves interplay between malignant and accessory cell types. Here, we performed a cellular and molecular comparison of the bone microenvironment in mouse models representing either metastatic indolence or growth, to identify mechanisms regulating cancer cell survival and fate. In vivo, we show that regardless of their fate, breast cancer cells in bone occupy niches rich in osteoblastic cells. As the number of osteoblasts in bone declines, so does the ability to sustain large numbers of breast cancer cells and support metastatic outgrowth. In vitro, osteoblasts protected breast cancer cells from death induced by cell stress and signaling via gap junctions was found to provide important juxtacrine protective mechanisms between osteoblasts and both MDA-MB-231 (TNBC) and MCF7 (ER+) breast cancer cells. Combined with mathematical modelling, these findings indicate that the fate of DTCs is not controlled through the association with specific vessel subtypes. Instead, numbers of osteoblasts dictate availability of protective niches which breast cancer cells can colonize prior to stimulation of metastatic outgrowth.

Signaling Pathway Mediating Myeloma Cell Growth and Survival.

Simple SummaryThe bone marrow (BM) microenvironment plays a crucial role in pathogenesis of multiple myeloma (MM), and delineation of the intracellular signaling pathways activated in the BM microenvironment in MM cells is essential to develop novel therapeutic strategies to improve patient outcome.The multiple myeloma (MM) bone marrow (BM) microenvironment consists of different types of accessory cells. Both soluble factors (i.e., cytokines) secreted from these cells and adhesion of MM cells to these cells play crucial roles in activation of intracellular signaling pathways mediating MM cell growth, survival, migration, and drug resistance. Importantly, there is crosstalk between the signaling pathways, increasing the complexity of signal transduction networks in MM cells in the BM microenvironment, highlighting the requirement for combination treatment strategies to blocking multiple signaling pathways.

Biallelic Loss of BCMA Triggers Resistance to Anti-BCMA CAR T Cell Therapy in Multiple Myeloma

Biallelic Loss of BCMA Triggers Resistance to Anti-BCMA CAR T Cell Therapy in Multiple Myeloma

Transcriptional repression specifies the central cell for double fertilization

Double fertilization is a key innovation for the evolutionary success of angiosperms by which the two fertilized female gametes, the egg cell and central cell, generate the embryo and endosperm, respectively. The female gametophyte (embryo sac) enclosed in the sporophyte is derived from a one-celled haploid cell lineage. It undergoes successive events of mitotic divisions, cellularization, and cell specification to give rise to the mature embryo sac, which contains the two female gametes accompanied by two types of accessory cells, namely synergids and antipodals. How the cell fate of the central cell is specified has long been equivocal and is further complicated by the structural diversity of female gametophyte across plant taxa. Here, MADS-box protein AGL80 was verified as a transcriptional repressor that directly suppresses the expression of accessory cell-specific genes to specify the central cell. Further genetic rescue and phylogenetic assay of the AGL80 orthologs revealed a possible conserved mechanism in the Brassicaceae family. Results from this study provide insight into the molecular determination of the second female gamete cell in Brassicaceae.

Stem Cell-Derived Exosomes as Nanotherapeutics for Autoimmune and Neurodegenerative Disorders.

To dissect therapeutic mechanisms of transplanted stem cells and develop exosome-based nanotherapeutics in treating autoimmune and neurodegenerative diseases, we assessed the effect of exosomes secreted from human mesenchymal stem cells (MSCs) in treating multiple sclerosis using an experimental autoimmune encephalomyelitis (EAE) mouse model. We found that intravenous administration of exosomes produced by MSCs stimulated by IFNγ (IFNγ-Exo) (i) reduced the mean clinical score of EAE mice compared to PBS control, (ii) reduced demyelination, (iii) decreased neuroinflammation, and (iv) upregulated the number of CD4+CD25+FOXP3+ regulatory T cells (Tregs) within the spinal cords of EAE mice. Co-culture of IFNγ-Exo with activated peripheral blood mononuclear cells (PBMCs) cells in vitro reduced PBMC proliferation and levels of pro-inflammatory Th1 and Th17 cytokines including IL-6, IL-12p70, IL-17AF, and IL-22 yet increased levels of immunosuppressive cytokine indoleamine 2,3-dioxygenase. IFNγ-Exo could also induce Tregs in vitro in a murine splenocyte culture, likely mediated by a third-party accessory cell type. Further, IFNγ-Exo characterization by deep RNA sequencing suggested that IFNγ-Exo contains anti-inflammatory RNAs, where their inactivation partially hindered the exosomes potential to induce Tregs. Furthermore, we found that IFNγ-Exo harbors multiple anti-inflammatory and neuroprotective proteins. These results not only shed light on stem cell therapeutic mechanisms but also provide evidence that MSC-derived exosomes can potentially serve as cell-free therapies in creating a tolerogenic immune response to treat autoimmune and central nervous system disorders.

T Helper Cell Cytokines Modulate Intestinal Stem Cell Renewal and Differentiation

In the small intestine, a niche of accessory cell types supports the generation of mature epithelial cell types from intestinal stem cells (ISCs). It is unclear, however, if and how immune cells in the niche affect ISC fate or the balance between self-renewal and differentiation. Here, we use single-cell RNA sequencing (scRNA-seq) to identify MHC class II (MHCII) machinery enrichment in two subsets of Lgr5+ ISCs. We show that MHCII+ Lgr5+ ISCs are non-conventional antigen-presenting cells in co-cultures with CD4+ T helper (Th) cells. Stimulation of intestinal organoids with key Th cytokines affects Lgr5+ ISC renewal and differentiation in opposing ways: pro-inflammatory signals promote differentiation, while regulatory cells and cytokines reduce it. Invivo genetic perturbation of Th cells or MHCII expression on Lgr5+ ISCs impacts epithelial cell differentiation and IEC fate during infection. These interactions between Th cells and Lgr5+ ISCs, thus, orchestrate tissue-wide responses to external signals.

Fate mapping during regeneration: Cells that undergo compensatory proliferation in damaged Drosophila eye imaginal discs differentiate into multiple retinal accessory cell types

Regeneration of tissues that have been damaged by cell loss requires new growth, often via proliferation of precursor cells followed by differentiation to replace loss of specific cell types. When regeneration occurs after normal differentiation of the tissue is complete, developmental pathways driving differentiation must be re-activated. How proliferation and differentiation are induced and balanced during regeneration is not well understood. To investigate these processes, we utilized a paradigm for tissue damage and regeneration in the developing Drosophila melanogaster eye. Previous studies have demonstrated that tissue damage resulting from extensive cell death stimulates quiescent, undifferentiated cells in the developing larval eye to re-enter the cell cycle and proliferate. Whether these cells are restricted to certain fates or can contribute to all retinal cell types and thus potentially be fully regenerative is not known. Here we found by fate mapping experiments that these cells are competent to differentiate into all accessory cell types in the retina but do not differentiate into photoreceptors, likely because cell cycle re-entry in response to damage occurs after photoreceptor differentiation has completed. We conclude that the ability to re-enter the cell cycle in response to tissue damage in the developing Drosophila eye is not restricted to precursors of a specific cell type and that cell cycle re-entry following damage does not disrupt developmental programs that control differentiation.

Highlights of This Issue

Ibrutinib is an irreversible tyrosine kinase inhibitor that targets Bruton's tyrosine kinase (BTK) as well as ErbB family kinases. Chen and colleagues found that ibrutinib potently inhibited the activity of ErbB kinases and the growth of HER2 overexpressing cells similarly as ErbB-targetted inhibitors in clinic. Compare to ErB inhibitors, the dual activity toward TEC and ErbB family kinases was unique to ibrutinib. In addition, it was shown that the anti-tumor effects of ibrutinib in breast cancer models are at clinically achievable levels. Since TEC kinases modulate several types of tumor accessory cells, the utility of ibrutinib's unique inhibitory spectrum is worthy of further exploration.Advanced/metastatic gastrointestinal stromal tumors (GIST), driven mainly by KIT or PDGFRA mutations, are treated with tyrosine kinase inhibitors (TKI) such as imatinib. However, the development of resistance remains to be a major clinical challenge. Gebreyohannes, Schöffski and colleagues showed the in vivo activity of cabozantinib, a novel small molecule multi-targeted TKI, in three GIST patient-derived xenograft models, characterized by different KIT mutations and sensitivity to standard TKI. Cabozantinib decreased or stabilized tumor burden, mainly through the inhibition of cell proliferation and tumor vascularization. Moreover, the effect observed was significantly better than the one obtained with imatinib.T cell immunoglobulin mucin-1 (TIM-1) is highly up-regulated in renal cell carcinoma and ovarian clear cell carcinoma. To exploit the expression of TIM-1 on these malignancies, the Celldex Therapeutics team developed CDX-014, which is an antibody drug conjugate comprised of a fully-human monoclonal antibody directed against an epitope of TIM-1, conjugated to the cytotoxic agent MMAE. Preclinical studies with this ADC demonstrated in vitro and in vivo efficacy against a variety of TIM-1 expressing cell lines and tumor models. With the successful completion of IND-enabling studies, activities are on-going to advance CDX-014 in clinical studies in renal cell carcinomas and other TIM-1 expressing tumors.122 anti-cancer agents were profiled in proliferation assays with cancer cell lines from diverse tumor tissues. For 38 compounds this is the first comparative profiling study. Good correlations were observed with other cell panels. Curve fitting led to a large source of variation. Hierarchical clustering revealed common mechanisms of action. Nine clusters, including EZH2, BET and TTK inhibitors, have not been reported before. The lack of clustering of several Aurora and PI3K inhibitors could be explained by differences in biochemical selectivity. The study shows that cell line profiling is an excellent tool for the unbiased classification of anti-proliferative compounds.

Phenotypical, Functional and Genetic Characterization of Mesenchymal Stem Cells Derived from the Spleen of Patients with Myelofibrosis

Splenic extramedullary hematopoiesis is a major clinico-pathological feature of patients with myelofibrosis. As in the bone marrow (BM), hematopoiesis in the spleen occurs thank to the interplay of hematopoietic progenitor cells with the microenvironment, which provides the regulatory mechanism for their differentiation, proliferation and trafficking. Among other components, such as vessels and extra-cellular matrix proteins, this microenvironment encompasses different types of accessory cells, including mesenchymal stromal cells (MSCs). We have recently reported that MSCs from the BM of patients with myelofibrosis harbor genetics abnormalities and display an altered functional activity, suggesting that a primary MSC defect may either lead to or favor the pathogenesis of the disease. Here, we describe the phenotypical, functional, and genetic profile of MSCs isolated from the spleen of 23 patients with myelofibrosis, who underwent splenectomy for anemia and/or for excessive size of the spleen, and compare them to splenic mesenchymal stromal cells (s-MSCs) from 7 healthy subjects (HSs) who were splenectomized following traumatic lesion. The study was approved by the institutional review board of IRCSS Policlinico San Matteo Foundation; patients and HSs gave written informed consent for participating to the study. Mononuclear cells (MNCs) were obtained by dissociation of small spleen fragments by means of the GentleMacs Dissociator device (Miltenyi Biotech, Germany), and s-MSCs were isolated and expanded according to the standard procedures used for BM-MSCs. S-MSCs were obtained in 9/23 patients and in 3/7 HSs and displayed no significant differences for morphology and differentiation ability into adipocytic and osteoblastic lineages. However, the clonogenic efficiency of s-MSCs from patients with myelofibrosis was statistically higher than that of HSs (0.07 colonies/106 MNCs, range 0.03-0.01, vs 0.03/106 MNCs, range 0.03-0.04, respectively; p=0.048), whereas doubling time and time to senescence were not statistically different. Flow cytometric assessment of standard surface antigens (CD13, CD14, CD34, CD45, CD73, CD90, CD105) confirmed the mesenchymal nature of the cells grown in the cultures, and was similar between patients’ and HSs’ s-MSCs. When nestin expression was determined, no significant differences in the frequency of MSCs expressing this antigen was observed; however, nestin Mean Fluorescence Intensity (MFI) of patients’ s-MSCs was significantly lower than that of s-MSCs from HSs (22, range 6-45, vs 97, range 65-100, respectively; p=0.035). Patients’ s-MSCs also displayed a reduced capacity to sustain long term hematopoiesis in vitro in a classical Long Term Culture-Initiating Cell assay. However, when normal cord blood-derived CD34+ cells were co-cultured onto patients’ s-MSCs in a transwell system for 13 days, the output of CD41+ megakaryocytic cells increased with respect to culture where CD34+ cells were plated onto HSs’ s-MSCs [21,5% vs 14,2% w/o recombinant human thrombopoietin (rhTPO), respectively, p=0,043; 60,2% vs 33,6% with rhTPO, respectively, p=0,01] at detriment of CD33+ cells (41,5% vs 48,6% w/o rhTPO, respectively, p=0,049; 10,4% vs 29,4% with rhTPO, respectively, p=0,012]. Finally, an abnormal karyotype [46XXt(5;17)(4-12)] was detected in 1 out of 18 metaphases of 1 out of 3 patient s-MSCs, while a normal karyotype was always observed in 2 out of 2 HSs’ s-MSC. This extensive characterization of s-MSCs shows that s-MSCs of patients with myelofibrosis display functional and genetic abnormalities compared to those isolated from HSs. The low level of nestin expression suggests that the hematopoietic niche of the spleen of patients with myelofibrosis can be defective and responsible for the increased trafficking of CD34+ cells that is observed in these patients, whereas the increased differentiation into the megakaryocytic lineage indicates a role of the splenic niche in leading hematopoiesis toward a pathological profile. All together, our data suggest that s-MSCs play a role in the pathogenesis of myelofibrosis and could be, therefore, a potential target for the treatment of the disease. DisclosuresNo relevant conflicts of interest to declare.

Advanced imaging and tissue engineering of the human limbal epithelial stem cell niche.

The limbal epithelial stem cell niche provides a unique, physically protective environment in which limbal epithelial stem cells reside in close proximity with accessory cell types and their secreted factors. The use of advanced imaging techniques is described to visualize the niche in three dimensions in native human corneal tissue. In addition, a protocol is provided for the isolation and culture of three different cell types, including human limbal epithelial stem cells from the limbal niche of human donor tissue. Finally, the process of incorporating these cells within plastic compressed collagen constructs to form a tissue-engineered corneal limbus is described and how immunohistochemical techniques may be applied to characterize cell phenotype therein.

The effects of CA IX catalysis products within tumor microenvironment

Solid tumors are composed of both cancer cells and various types of accessory cells, mainly fibroblasts, that collectively compose the so called tumor-microenvironment. Cancer-associated fibroblasts have been described to actively participate in cancer progression by establishing a cytokine-mediated as well as metabolic crosstalk with cancer cells. In the present paper we show that activated human fibroblasts are able to boost tumor cells proliferation and that this effect is greatly dependent on stromal carbonic anhydrase IX (CA IX) activity. In fact fibroblasts show a strong upregulation of CA IX expression upon activation by cancer cells, while CA IX products, protons and bicarbonate, exert differential effects on cancer cells proliferation. While acidification of extracellular pH, a typical condition of rapidly growing solid tumors, is detrimental for tumor cells proliferation, bicarbonate, through its organication, supplies cancer cells with intermediates useful to sustain their high proliferation rate. Here we propose a new kind of fibroblasts/tumor cells crosstalk within tumor microenvironment, mediated by stromal CA IX products, aimed to favor cancer cells growth, opening new perspectives on CA IX role in tumor microenvironment.

Pretreatment with TCDD exacerbates liver injury from Concanavalin A: critical role for NK cells.

For many liver diseases, including viral and autoimmune hepatitis, immune cells play an important role in the development and progression of liver injury. Concanavalin A (Con A) administration to rodents has been used as a model of immune-mediated liver injury resembling human autoimmune hepatitis. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been demonstrated to alter the development of immune-mediated diseases. Mice pretreated with TCDD developed exacerbated liver injury in response to administration of a mild dose (6 mg/kg) of Con A. In the present study, we tested the hypothesis that TCDD pretreatment exacerbates Con A-induced liver injury by enhancing the activation and recruitment of accessory cell types including neutrophils, macrophages, and natural killer (NK) cells. Mice were treated with 0, 0.3, 3, or 30 μg/kg TCDD and 4 days later with Con A or saline. TCDD pretreatment with doses of 3 and 30 μg/kg significantly increased liver injury from Con A administration. The plasma concentrations of neutrophil chemokines were significantly increased in TCDD-pretreated mice after Con A administration. NKT cell-deficient (CD1d KO) mice were used to examine whether NKT cells were required for TCDD/Con A-induced liver injury. CD1d KO mice were completely protected from liver injury induced by treatment with Con A alone, whereas the injury from TCDD/Con A treatment was reduced but not eliminated. However, T-cell deficient (RAG1 KO) mice were protected from liver injury induced by Con A irrespective of pretreatment with TCDD. TCDD/Con A treatment increased the percentage of NK cells expressing the activation marker CD69. Depletion of NK cells prior to treatment resulted in significant reductions in plasma interferon-γ and liver injury from TCDD/Con A treatment. In summary, exposure to TCDD exacerbated the immune-mediated liver injury induced by Con A, and our findings suggest that NK cells play a critical role in this response.

Osteogenesis and expression of the bone marrow niche in endothelial cell‐depleted HipOPs

The identification and purification of murine multipotent mesenchymal stem cells (MSCs) have been difficult due to their low frequency, the presence of contaminating cell types and lack of unambiguous markers. Using a magnetic micro-beads negative selection technique to remove hematopoietic cells from mouse bone marrow stromal cells (BMSCs), our lab recently isolated a highly purified osteoprogenitor (HipOP) population that was also enriched for other mesenchymal precursors, including MSCs [Itoh and Aubin, 2009]. We now report that HipOPs are also highly enriched in vascular endothelial cells (VECs), which we hypothesized were an accessory cell type regulating osteogenesis. However, when VECs were immunodepleted from HipOPs with anti-CD31 antibodies, the resulting CD31(-) HipOP population had equal osteogenic capacity to the HipOPs in vitro and in vivo. Analysis of gene expression of Ncad, Pth1r, Ang1, Cxcl12, Jag1, Pdgfr-β, α-sma, Desmin, and Ng2 suggested that both HipOPs and CD31(-) HipOPs are hemopoietic stem cell (HSC) niche populations. However, the data support the view that osteoblast differentiation and depletion of VECs modulate the HSC niche.

Cognate Antigen Stimulation Generates Potent CD8+ Inflammatory Effector T Cells

Inflammatory reactions are believed to be triggered by innate signals and have a major protective role by recruiting innate immunity cells, favoring lymphocyte activation and differentiation, and thus contributing to the sequestration and elimination of the injurious stimuli. Although certain lymphocyte types such as TH17 cells co-participate in inflammatory reactions, their generation from the naïve pool requires the pre-existence of an inflammatory milieu. In this context, inflammation is always regarded as beginning with an innate response that may be eventually perpetuated and amplified by certain lymphocyte types. In contrast, we here show that even in sterile immunizations or in MyD88-deficient mice, CD8 T cells produce a burst of pro-inflammatory cytokines and chemokines. These functions follow opposite rules to the classic CD8 effector functions since they are generated prior to cell expansion and decline before antigen elimination. As few as 56 CD8+ inflammatory effector cells in a lymph node can mobilize 107 cells in 24 h, including lymphocytes, natural killer cells, and several accessory cell types involved in inflammatory reactions. Thus, although inflammation modulates cognate responses, CD8 cognate responses also initiate local inflammatory reactions.

Accessory Cells From Diverse Tissue Microenvironments Alter Tumor Cell Responses to Multitargeted Kinase Inhibitors: Therapeutic Implications From a Large-Scale Systematic Evaluation.

Abstract 2469 Introduction:Kinases are integral components of diverse signaling cascades which can mediate microenvironment-derived resistance of tumor cells to apoptosis. We thus hypothesized that tumor cell-microenvironment interactions alter the anti-tumor activity of multi-targeted kinase inhibitors and could contibute to discordant efficacy results in conventional preclinical models which do not account for these interactions vs. in clinical studies. Methods:We evaluated 6 multi-targeted kinase inhibitors initially developed to target IGF1R (XL-228), Akt/p70S6K (EXEL-6075), B-/C-RAF(EXEL-0094), CHK1/2 (XL844), met/VEGFR2(EXEL-6323) and JAK2 (XL019). Compounds were tested (0–20 μM, 48 hrs) against luciferase-expressing human multiple myeloma (MM) (n=8), mantle cell lymphoma (n=2) and breast cancer (n=1) cell lines. For each of these treatments, tumor cells were cultured in isolation or co-cultured with immortalized human bone marrow stromal cells (BMSCs, n=2); lung fibroblasts; hepatocytes; or brain astrocytes. Tumor cell viability was quantified by tumor cell compartment-specific bioluminescence imaging (CS-BLI). For each compound and tumor cell line, the area under the dose-response curve (AUC) was calculated as % of the AUC that corresponds to 100% tumor cell viability at all doses, to provide cumulative measure of drug activity across the range of tested drug concentrations. Differences in % AUCs in presence vs. absence of accessory cells were compared by 2-tailed paired t-test. The average value and skewness of distribution for log(10)IC50 of enzymatic activity for each kinase and inhibitor tested were calculated for quantitative comparison of multi-targeted nature of these compounds. Results:Co-culture of most tumor cell lines with accessory cells triggered resistance for 5 of 6 compounds tested, as evidenced by statistically significant (p<0.001 for all tests) increases in %AUC with vs. without co-culture with accessory cells (average %AUC differences of 4.7%, 23.4%, 6.2%, 9.7% and 10.8% for IGF1R, Akt/p70S6K, B-/C-RAF, CHK1/2; and met/VEGFR2 inhibitors, respectively, with corresponding 95% C.I. of 2.0–7.5%, 18.2–28.6%, 2.0–10.4%, 5.3–14.1%, and 3.9–17.6%). In contrast, co-culture with accessory cells increased the response of most tumor cell lines to JAK2 inhibitor (average AUC decrease of 13.8%, 95%CI 1.9–16.4, p<0.001). Results were consistent and statistically significant when analysis was restricted to MM cell lines only. For some inhibitors, heterogeneous responses of different tumor cells to accessory cells were noted. For example, for met/VEGFR2 and CHK1/2 inhibitors, 58.0% (40/69) and 60.0% (36/60) of experimental conditions, respectively, showed co-culture-induced resistance, while 31.9% (22/69) and 18.0% (11/60) of cocultures, respectively, showed sensitization to these inhibitors. High frequency of sensitization was observed in co-cultures of a bortezomib/Dex-resistant subline of MM1S cells treated with met/VEGFR2 inhibitor (63.6%, 7/11 of co-cultures tested), and in co-cultures with BMSCs treated with CHK1/2 inhibitor (29.6% of co-cultures tested). The degree of sensitization or resistance by accessory cells (%AUC difference with vs. without co-culture) did not correlate with quantitative measures of multi-targeted nature for each inhibitor. Conclusions:Accessory cells from different tissues significantly modulated tumor cell responses to diverse multi-targeted kinase inhibitors. Co-cultures enhanced the anti-tumor activity of JAK2 inhibitor, but decreased the activity of the other compounds tested. These different responses did not correlate with how multitargeted each inhibitor was. The sensitization to the JAK inhibitor may specifically reflect increased dependency on JAK signaling for tumor cells when they interact with accessory cells. Accessory cell-induced resistance to some kinase inhibitors (e.g. IGF1R, Akt, CHK1–2) may account for observed differences in their preclinical vs. clinical efficacy. These results provide a rationale for preclinical testing of compounds against large panels of tumor cell lines and clinically-relevant accessory cell types to help develop therapeutics against tumors localized in specific tissue microenvironmental niches. Disclosures:McMillin:Axios Biosciences: Equity Ownership. Mitsiades:Millennium Pharmaceuticals: Honoraria; Celgene: Honoraria; Novartis Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Merck &Co.: Honoraria; Centocor: Honoraria; Arno Therapeutics: Honoraria; Amgen: Research Funding; AVEO Pharma: Research Funding; OSI: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Johnson & Johnson: Research Funding; PharmaMar: Licensing royalties Other; Axios Biosciences: Uncompensated Role as advisor, Uncompensated Role as advisor Other.