Nupur Bhattacharya

An interactive reference framework for modeling a dynamic immune system

Single-cell measurements map immunity Multiple characteristics of individual cells define cell types and their physiological states. Spitzer et al. quantitated the abundance of 39 different cell surface proteins or transcription factors on individual cells of the mouse immune system. They used these measurements to create a map that clustered similar individual cells into groups corresponding to cell type and function. Their extensible experimental platform will allow the inclusion of other data types and data from independent laboratories. Science, this issue 10.1126/science.1259425 Cytometry meets mass spectrometry to create a functional map of the immune system. INTRODUCTION Immune cells constitute an interacting hierarchy that coordinates its activities according to genetic and environmental contexts. This systemically mobile network of cells results in emergent properties that are derived from dynamic cellular interactions. Unlike many solid tissues, where cells of given functions are localized into substructures that can be readily defined, the distribution of phenotypically similar immune cells into various organs complicates discerning any modest differences between them. Over decades of investigation into immune functions during health and disease, research has necessarily focused on understanding the individual cell types within the immune system, and, more recently, toward identifying interacting cells and the messengers they use to communicate. RATIONALE Methods of single-cell analysis, such as flow cytometry, have led the effort to enumerate and quantitatively characterize immune cell populations. As research has accelerated, our understanding of immune organization has surpassed the technical limitations of fluorescence-based flow cytometry. With the advent of mass cytometry, which enables measuring significantly more features of individual cells, most known immune cell types can now be identified from within a single experiment. Leveraging this capability, we set out to initiate an immune system reference framework to provide a working definition of immune organization and enable the integration of new data sets. RESULTS To build a reference framework from mass cytometry data, we developed a novel algorithm to transform the single-cell data into intuitive maps. These Scaffold maps provide a data-driven interpretation of immune organization while also integrating conventional immune cell populations as landmarks to orient the user. By applying Scaffold maps to data from the bone marrow of wild-type C57BL/6 mice, the method reconstructed the organization within this complex developmental organ. Using this sample as a reference point, the unique organization of immune cells within various organs across the body was revealed. The maps recapitulated canonical cellular phenotypes while revealing reproducible, tissue-specific deviations. The approach revealed influences of genetic variation and circadian rhythms on immune structure, permitted direct comparisons of murine and human blood cell phenotypes, and even enabled archival fluorescence-based flow cytometry data to be mapped onto the reference framework. CONCLUSION This foundational reference map provides a working definition of systemic immune organization to which new data can be integrated to reveal deviations driven by genetics, environment, or pathology. Beyond providing an analytical framework to understand immune organization from the unified data set generated here, the approaches we describe can serve as a data repository for collating experimental data from the research community, including gene expression and mutational analysis. Efforts that characterize cellular behavior in this open-source approach will continue to improve upon the initiating reference presented here to reveal the inherent structure in biological networks of immunity for clinical benefit. Building a dynamic immune system reference framework. By combining mass cytometry with the Scaffold maps algorithm, the cellular organization of any complex sample can be transformed into an intuitive and interactive map for further analysis. By first choosing one foundational sample as a reference (i.e., the bone marrow of wild-type mice), the effects of any perturbation can be readily identified in this framework. Immune cells function in an interacting hierarchy that coordinates the activities of various cell types according to genetic and environmental contexts. We developed graphical approaches to construct an extensible immune reference map from mass cytometry data of cells from different organs, incorporating landmark cell populations as flags on the map to compare cells from distinct samples. The maps recapitulated canonical cellular phenotypes and revealed reproducible, tissue-specific deviations. The approach revealed influences of genetic variation and circadian rhythms on immune system structure, enabled direct comparisons of murine and human blood cell phenotypes, and even enabled archival fluorescence-based flow cytometry data to be mapped onto the reference framework. This foundational reference map provides a working definition of systemic immune organization to which new data can be integrated to reveal deviations driven by genetics, environment, or pathology.

Epigenetic Upregulation of lncRNAs at 13q14.3 in Leukemia Is Linked to the In Cis Downregulation of a Gene Cluster That Targets NF-kB

Non-coding RNAs are much more common than previously thought. However, for the vast majority of non-coding RNAs, the cellular function remains enigmatic. The two long non-coding RNA (lncRNA) genes DLEU1 and DLEU2 map to a critical region at chromosomal band 13q14.3 that is recurrently deleted in solid tumors and hematopoietic malignancies like chronic lymphocytic leukemia (CLL). While no point mutations have been found in the protein coding candidate genes at 13q14.3, they are deregulated in malignant cells, suggesting an epigenetic tumor suppressor mechanism. We therefore characterized the epigenetic makeup of 13q14.3 in CLL cells and found histone modifications by chromatin-immunoprecipitation (ChIP) that are associated with activated transcription and significant DNA-demethylation at the transcriptional start sites of DLEU1 and DLEU2 using 5 different semi-quantitative and quantitative methods (aPRIMES, BioCOBRA, MCIp, MassARRAY, and bisulfite sequencing). These epigenetic aberrations were correlated with transcriptional deregulation of the neighboring candidate tumor suppressor genes, suggesting a coregulation in cis of this gene cluster. We found that the 13q14.3 genes in addition to their previously known functions regulate NF-kB activity, which we could show after overexpression, siRNA–mediated knockdown, and dominant-negative mutant genes by using Western blots with previously undescribed antibodies, by a customized ELISA as well as by reporter assays. In addition, we performed an unbiased screen of 810 human miRNAs and identified the miR-15/16 family of genes at 13q14.3 as the strongest inducers of NF-kB activity. In summary, the tumor suppressor mechanism at 13q14.3 is a cluster of genes controlled by two lncRNA genes that are regulated by DNA-methylation and histone modifications and whose members all regulate NF-kB. Therefore, the tumor suppressor mechanism in 13q14.3 underlines the role both of epigenetic aberrations and of lncRNA genes in human tumorigenesis and is an example of colocalization of a functionally related gene cluster.

Allogeneic IgG combined with dendritic cell stimuli induce antitumour T-cell immunity

Whereas cancers grow within host tissues and evade host immunity through immune-editing and immunosuppression, tumours are rarely transmissible between individuals. Much like transplanted allogeneic organs, allogeneic tumours are reliably rejected by host T cells, even when the tumour and host share the same major histocompatibility complex alleles, the most potent determinants of transplant rejection. How such tumour-eradicating immunity is initiated remains unknown, although elucidating this process could provide the basis for inducing similar responses against naturally arising tumours. Here we find that allogeneic tumour rejection is initiated in mice by naturally occurring tumour-binding IgG antibodies, which enable dendritic cells (DCs) to internalize tumour antigens and subsequently activate tumour-reactive T cells. We exploited this mechanism to treat autologous and autochthonous tumours successfully. Either systemic administration of DCs loaded with allogeneic-IgG-coated tumour cells or intratumoral injection of allogeneic IgG in combination with DC stimuli induced potent T-cell-mediated antitumour immune responses, resulting in tumour eradication in mouse models of melanoma, pancreas, lung and breast cancer. Moreover, this strategy led to eradication of distant tumours and metastases, as well as the injected primary tumours. To assess the clinical relevance of these findings, we studied antibodies and cells from patients with lung cancer. T cells from these patients responded vigorously to autologous tumour antigens after culture with allogeneic-IgG-loaded DCs, recapitulating our findings in mice. These results reveal that tumour-binding allogeneic IgG can induce powerful antitumour immunity that can be exploited for cancer immunotherapy.

Lenalidomide treatment of chronic lymphocytic leukaemia patients reduces regulatory T cells and induces Th17 T helper cells

Chronic lymphocytic leukaemia (CLL) remains an incurable disease, and the development of novel treatment options is therefore imperative. The immunomodulatory drug lenalidomide has shown promise in early clinical trials (Chanan-Khan & Porter, 2006; Chanan-Khan & Cheson, 2008; Ferrajoli et al, 2008). However, the exact mode of action of lenalidomide is unclear, and development of a ‘tumour flare reaction’ (TFR) is a critical side effect (Andritsos et al, 2008). In order to understand the mode of action of lenalidomide in CLL, we performed an analysis of intra-patient follow-up samples to assess the potential in vivo target cell population that contributes to effects and side effects. We initially investigated whether correlations that were previously reported (Andritsos et al, 2008) between in vitro changes of surface markers on CLL cells and development of TFR could also be found in vivo. We analysed peripheral blood lymphocytes (PBL) from three patients (Table SI) that were treated with lenalidomide (5 mg/d starting dose, escalated by 5 mg/d every 28 d). Two of the patients developed TFR upon therapy (Fig 1). However, the most pronounced upregulation of CD86 and CD40 in vivo was detected in Patient 2 who did not experience a TFR (Fig 2A), while Patient 1 (with a TFR) showed no upregulation of CD40. These findings would suggest that there is no strict correlation of development of a TFR with in vivo induction of CD40 and/or CD86 on CLL cells. This is in contrast to effects of lenalidomide in vitro, which have also been reported previously (Andritsos et al, 2008): treatment resulted in upregulation of CD40 and CD86 in the majority of samples (9/12 and 11/12, respectively, Figure S1). Interestingly this effect was also observed directly on pure CD19 sorted cells (8/12 and 11/12), excluding indirect induction by non-malignant bystander cells (Figure S1). In contrast, only 30–58% of patients experience a TFR after treatment in vivo, depending on lenalidomide dosage (Chanan-Khan & Porter, 2006; Ferrajoli et al, 2008). This makes it unlikely that in vitro upregulation of CD40 and CD86 predicts TFR. Also, in contrast to previous in vitro analyses (Lapalombella et al, 2008), in vivo only one of the patients presented with more CD38 cells during treatment (Patient 1, Fig 2A). A lymph node (LN) aspirate obtained from Patient 3 during TFR however showed twofold more CD38 CLL cells than cells drawn from PBL (data not shown), but no LN aspirate from a timepoint before initiation of treatment was available for comparison. Thus, our findings uncovered interesting differences between the in vitro and in vivo effects of lenalidomide on CLL cells in a small number of patients. Similar to thalidomide, lenalidomide is postulated to impact not only on CLL cells, but also activate and induce proliferation of T cells and natural killer (NK) cells in a mouse model (Hernandez-Ilizaliturri et al, 2005), in healthy volunteers and in patients suffering from multiple myeloma (Chang et al, 2006) and activate them for antibody-dependent cellular cytotoxicity (ADCC) against CLL cells in vitro (Lapalombella et al, 2008). Similarly, we found induction of CD16CD56NK cell numbers in 2/3 CLL patients after treatment with lenalidomide in vivo (Fig 2B). At the same time, the activation status of NK cells decreased as measured by CD69 and CD25 levels. However, it has to be kept in mind that these are early

Detection of Intestinal Cancer by Local, Topical Application of a Quenched Fluorescence Probe for Cysteine Cathepsins

Early detection of colonic polyps can prevent up to 90% of colorectal cancer deaths. Conventional colonoscopy readily detects the majority of premalignant lesions, which exhibit raised morphology. However, lesions that are flat and depressed are often undetected using this method. Therefore, there is a need for molecular-based contrast agents to improve detection rates over conventional colonoscopy. We evaluated a quenched fluorescent activity-based probe (qABP; BMV109) that targets multiple cysteine cathepsins that are overexpressed in intestinal dysplasia in a genetic model of spontaneous intestinal polyp formation and in a chemically induced model of colorectal carcinoma. We found that the qABP selectively targets cysteine cathepsins, resulting in high sensitivity and specificity for intestinal tumors in mice and humans. Additionally, the qABP can be administered by either intravenous injection or by local delivery to the colon, making it a highly valuable tool for improved detection of colorectal lesions using fluorescence-guided colonoscopy.

Chronic lymphocytic leukemia and 13q14: miRs and more

Loss of a critical region in 13q14.3 [del(13q)] is the most common genomic aberration in chronic lymphocytic leukemia (CLL), occurring in more than 50% of patients (Stilgenbauer et al., Oncogene 1998;16:1891 – 1897, Dohner et al., N Engl J Med 2000;343:1910 – 1916). Despite extensive investigations, no point mutations have been found in the remaining allele that would inactivate one of the candidate tumor suppressor genes and explain the pathomechanism postulated for this region. However, the genes in the region are significantly down-regulated in CLL cells, more than would be expected by gene dosage, and recently a complex epigenetic regulatory mechanism was identified for 13q14.3 in non-malignant cells that involves asynchronous replication timing and monoallelic expression of candidate tumor suppressor genes. Here, we propose a model of a multigenic pathomechanism in 13q14.3, where several tumor suppressor genes, including the miRNA genes miR-16-1 and miR-15a, are co-regulated by the two long non-coding RNA genes DLEU1 and DLEU2 that span the critical region. Furthermore, we propose these co-regulated genes to be involved in the same molecular pathways, thereby also forming a functional gene cluster. Elucidating the molecular and cellular function of the 13q14.3 candidate genes will shed light on the underlying pathomechanism of CLL.

High-throughput detection of nuclear factor-kappaB activity using a sensitive oligo-based chemiluminescent enzyme-linked immunosorbent assay

Contemporary research on cellular signaling has undergone a shift of focus from qualitative measurements of single signaling pathways to high‐throughput quantitation of comprehensive signaling networks. Notably, nuclear factor‐kappaB (NFκB) is a family of transcription factors involved in immune and inflammatory responses, developmental processes, cellular growth and apoptosis and is deregulated in a number of disease states. We have established a chemiluminescent oligonucleotide‐based enzyme‐linked immunosorbent assay (co‐ELISA) that is simple and quantitative. In contrast to currently used assays, it allows quantitation of all NFκB components (i.e., RelA, p50, p52, RelB and c‐Rel). In addition, it can make use of whole extract and does not require cumbersome nuclear/cytosolic fractionation, saving time and resources. Co‐ELISA has a 3.5‐ to 43‐fold higher signal‐over‐noise ratio than currently available assays, whereas the percent relative standard deviation is 3‐ to 6‐fold lower. Furthermore, the novel method is faster than electrophoretic mobility shift assay, not restricted to transfectable cells as is the case for luciferase reporter assays and 10 times more cost efficient than commercially available ELISA assays. Co‐ELISA is a sensitive, fast and cost‐efficient quantitation method for all DNA‐binding NFκB proteins that can be used in high‐throughput experimentation.

Nurse-like cells show deregulated expression of genes involved in immunocompetence.

Chronic lymphocytic leukaemia (CLL) cells convert CD14+ cells from patients into ‘nurse‐like’ cells (NLCs). CLL cells can also convert CD14+ peripheral blood mononuclear cells (PBMCs) from healthy donors into cells with morphological similarities to NLCs (CD14CLL‐cells). However it is unclear whether only CLL cells induce this conversion process. This study showed that CD14+ PBMCs from healthy donors could also be converted into differentiated cells (CD14B‐cells) by non‐malignant B‐cells. In order to identify changes specifically induced by CLL cells, we compared gene expression profiles of NLCs, CD14CLL‐cells and CD14B‐cells. CD14+ cells cultured with CLL cells were more similar to NLCs than those cultured with non‐malignant B‐cells. The most significant changes induced by CLL cells were deregulation of the antigen presentation pathway and of genes related to immunity. NLCs had reduced levels of lysozyme activity, CD74 and HLA‐DR in‐vitro while expression of inhibitory FCGR2B was increased. These findings suggest an impaired immunocompetence of NLCs which, if found in‐vivo, could contribute to the immunodeficiency in CLL patients.

Spatial organization of dendritic cells within tumor draining lymph nodes impacts clinical outcome in breast cancer patients

BackgroundDendritic cells (DCs) are important mediators of anti-tumor immune responses. We hypothesized that an in-depth analysis of dendritic cells and their spatial relationships to each other as well as to other immune cells within tumor draining lymph nodes (TDLNs) could provide a better understanding of immune function and dysregulation in cancer.MethodsWe analyzed immune cells within TDLNs from 59 breast cancer patients with at least 5 years of clinical follow-up using immunohistochemical staining with a novel quantitative image analysis system. We developed algorithms to analyze spatial distribution patterns of immune cells in cancer versus healthy intra-mammary lymph nodes (HLNs) to derive information about possible mechanisms underlying immune-dysregulation in breast cancer. We used the non-parametric Mann–Whitney test for inter-group comparisons, Wilcoxon Matched-Pairs Signed Ranks test for intra-group comparisons and log-rank (Mantel-Cox) test for Kaplan Maier analyses.ResultsDegree of clustering of DCs (in terms of spatial proximity of the cells to each other) was reduced in TDLNs compared to HLNs. While there were more numerous DC clusters in TDLNs compared to HLNs,DC clusters within TDLNs tended to have fewer member DCs and also consisted of fewer cells displaying the DC maturity marker CD83. The average number of T cells within a standardized radius of a clustered DC was increased compared to that of an unclustered DC, suggesting that DC clustering was associated with T cell interaction. Furthermore, the number of T cells within the radius of a clustered DC was reduced in tumor-positive TDLNs compared to HLNs. Importantly, clinical outcome analysis revealed that DC clustering in tumor-positive TDLNs correlated with the duration of disease-free survival in breast cancer patients.ConclusionsThese findings are the first to describe the spatial organization of DCs within TDLNs and their association with survival outcome. In addition, we characterized specific changes in number, size, maturity, and T cell co-localization of such clusters. Strategies to enhance DC function in-vivo, including maturation and clustering, may provide additional tools for developing more efficacious DC cancer vaccines.

Non-malignant B cells and chronic lymphocytic leukemia cells induce a pro-survival phenotype in CD14 + cells from peripheral blood

Non-malignant B cells and chronic lymphocytic leukemia cells induce a pro-survival phenotype in CD14 + cells from peripheral blood