Distinct Leukocyte Subsets Research Articles

Insufficient PD-1 expression during active autoimmune responses: a deep single-cell proteomics analysis in inflammatory arthritis.

Programmed cell death protein-1 (PD-1) maintains peripheral immune tolerance by preventing T cell continuous activation. Aiming to understand the extent of PD-1 expression in inflammatory arthritis beyond its involvement with T cells, we assess its presence on various circulating single cells. Mass cytometry analysis of patients with active seropositive/seronegative rheumatoid (RA; n=9/8) and psoriatic (PsA; n=9) arthritis versus healthy controls (HC; n=13), re-evaluating patients after 3 months of anti-rheumatic treatment. PD-1 was expressed in all leukocyte subpopulations, with the highest PD-1+ cell frequencies in eosinophils (59-73%) and T cells (50-60%), and the lowest in natural-killer cells (1-3%). PD-1+ cell frequencies and PD-1 median expression were comparable between patient subgroups and HC, in the majority of cell subsets. Exceptions included increases in certain T cell/B cell subsets of seropositive RA and specific monocyte subsets and dendritic cells of PsA; an expanded PD-1+CD4+CD45RA+CD27+CD28+ T subset, denoting exhausted T cells, was common across patient subgroups. Strikingly, significant inverse correlations between individual biomarkers of systemic inflammation (ESR and/or serum CRP) and PD-1+ cell frequencies and/or median expression were evident in several innate and adaptive immunity cell subsets of RA and PsA patients. Furthermore, all inverse correlations noted in individuals with active arthritis were no longer discernible in those who attained remission/low disease activity post-treatment. PD-1 expression may be insufficient, relative to the magnitude of the concomitant systemic inflammatory response on distinct leukocyte subsets, varying between RA and PsA. Our results point to the potential therapeutic benefits of pharmacological PD-1 activation, to rebalance the autoimmune response and reduce inflammation.

Abstract TP295: Temporal Characterization of Immune Responses in the Rat Brain Following Ischemic Stroke Using In-Depth Flow Cytometry Panels

Introduction: Rat models are widely used in stroke research as they are well suited for behavior studies and brain imaging techniques. However, methods for studying immune responses and identifying distinct leukocyte subsets in rats lag behind mice in part due to lack of specific antibodies for flow cytometry. Several flow cytometry antibody panels have been used to study the immune response of the rat brain after stroke, but they did not distinguish between pro- and anti-inflammatory macrophages, which is critical for stroke-induced neuroinflammation research. Here, we developed flow cytometry panels to measure myeloid and lymphoid leukocyte subsets in rat brain, blood, and spleen, with an emphasis on macrophage subtypes, and validated them in an ischemic stroke model. Method: Adult male (8-12 wk old) Nude and Sprague Dawley (SD) rats were subjected to distal MCAO. Ipsi- and contralateral hemispheres, blood, and spleen were collected and analyzed by flow cytometry at different time points post-stroke. Cell populations were sorted using FACS and characterized using qPCR or morphological analysis to confirm our FACS gating strategy. Results: We discriminated microglia, 3 monocyte/macrophage subsets, granulocytes, dendritic cells, NK cells, B cells, and T cells in the brain and periphery. The temporal profile of leukocyte subsets after stroke resembled those observed in mice, validating the reliability of our antibody panels: granulocytes and monocytes peaked in the first 2-3 days post-stroke, B and T cells peaked around 4-5 days, followed by a gradual increase of ‘microglia’ over 14 days. Using a combination of CD43 and His48 to differentiate monocyte/macrophage subpopulations, we observed a shift in these subsets over time, akin to the shift from pro- to anti-inflammatory macrophages reported in stroke-injured mouse brains. Notably, Nude rats differed from the SD rats in terms of His48 expression; SD rat brain monocyte/macrophage population was predominantly His48lo, while Nude rats had both His48lo and His48hi subsets. Conclusion: Our flow cytometry panels for rats distinguished leukocyte subsets, including monocyte/macrophage subpopulations, providing a valuable tool to study the complex immune responses in rat models of neurological disorders.

Identification of novel genes that regulate B-1 cells

Abstract IgM is the first antibody produced in ontogeny and in response to an initial antigen exposure. It plays an important role in effective immune protection, and is required for maximal antigen-specific IgG responses. In mice, most circulating serum IgM is generated by fetal-derived B-1 cells in spleen and bone marrow. How B-1 cells preferentially develop into natural IgM secreting cells is incompletely understood. To identify candidate genes critical for B-1 cells development and/or maintenance we generated parts of a larger immunophenotyping database Knock Out Mouse Project (KOMP) providing results of the flow cytometric evaluation of spleen cells from 641 mouse strains, each containing a single-gene deletion as well as 200 individual congenic control mice, identifying 58 distinct leukocyte subsets for each spleen. We generated and curated a complete list of each gene found to be associated with B-1 cell changes. Seven genes were identified that significantly reduced frequencies of CD5+ B-1 cells in males and/or females, of which three were uniquely altering the B-1 cell subset. None of these genes have been associated with B-1 cell regulation previously. Using qRT-PCR we found that 4 showed B-1 cell intrinsic gene expression. Ongoing work is to determine the impact of each gene on serum antibody levels and immune protection to influenza virus challenge. This work was supported by NIH UM1 OD023221 (KCKL), R0AI148652 (NB), and the Graduate Student Support Program UC Davis (SR).

027 Memory B cells of atopic individuals preferentially express IL-31RA: a putative role of IL-31 in B cell biology

The novel cytokine IL-31 and its receptor have been shown to play a central role in bridging the immune system with neurons, epithelial surfaces and connective tissues. Although increasing evidence has demonstrated their role in inflammation, their involvement in the expansion of distinct leukocyte subsets is not fully understood. Detailed analyses of Il31-transgenic (Eμ-Lck) mice indicated that next to the induction of pruritus and skin lesions, mice also presented enlarged lymph nodes with increased B cell frequency. Therefore, we aimed at characterizing the role of IL-31 signaling in B cell biology. Using flow cytometry, the frequency of total B cells and specific B cell subsets was assessed in peripheral lymph nodes and bone marrow of Il31-transgenic (Eμ-Lck) mice and wild-type controls. Moreover, IL-31RA expression was analyzed on human B cell subsets from peripheral blood of healthy and atopic dermatitis donors. We observed a significantly increased frequency of CD19+ B cells in bone marrow and peripheral lymph nodes in Il31-transgenic mice. Detailed analyses of B cell subsets indicated that Il31-transgenic mice showed a higher frequency of plasmablasts in peripheral lymph nodes. In human, memory B cells represented the major population expressing IL-31RA within the B cell compartment. Furthermore, atopic dermatitis patients with elevated IgE levels (>1000 kU/l) showed a higher IL-31RA expression on memory B cells compared to healthy donors. This is of particular interest since DOCK8 acts as a negative regulator of IL-31. DOCK8 deficiency-related hyper-IgE syndrome presents with elevated serum IgE levels and severe atopic dermatitis with increased IL-31 expression. Taken together, these findings point to a novel role of IL-31 in B cell biology and an atopic IgE-producing phenotype.

Programmed cell death protein 1 (PD1) deficiency induces cardiac inflammation during baseline conditions and ischemia/reperfusion injury

Abstract Background The Programmed cell death protein 1 (PD1) immune checkpoint is densely expressed on cardiac endothelial cells. The increasing clinical application of immune checkpoint inhibitor therapy targeting PD1 for the treatment of advanced malignancies has revealed profound cardiovascular side effects that have recently paralleled in a preclinical model. However, the effects of PD1 deficiency during baseline conditions and myocardial injury are so far unknown. Purpose This study aims to assess the impact of PD1 deficiency on myocardia immunity, and to evaluate the relevance of PD1 signalling in cardiac disease. Methods C57BL/6J wild-type mice and C57BL/6J Pdcd1−/− mice were purchased and bred at the animal facility. In-vivo ischaemia/reperfusion (I/R) injury was applied to assess the response in cardiac injury. In brief, mice were anesthetised followed by lateral thoracotomy and ligation of the left coronary artery for 45 min. For flow cytometry, hearts were removed and subjected to an enzymatic digestion. Single cell solutions were stained with different antibody panels to assess cardiac immune cells and the expression of programmed cell death protein 1 ligand 1 (PDL1). Western blot was conducted after homogenization of snap-frozen heart tissue using specific primary and corresponding secondary antibodies. Immunofluorescence and conventional haematoxylin and eosin stain from 4 μm sections were used to visualize the distribution of PDL1 in cardiac tissue. Results While no overt cardiac phenotype was observed in Pdcd1−/− mice, a profound upregulation of pro-inflammatory cytokines was determined during baseline conditions, including interleukin (IL) 1α IL4, and extracellular-signal regulated kinase (ERK) 1/2. NADPH oxidase 1 (NOX1) which is involved in production of reactive oxygen species and endothelial injury response was downregulated in Pdcd1−/− mice. Following I/R injury, a significant decline in endothelial PDL1 expression was observed, which was attributed to changes in the area at risk, as shown by immunofluorescence staining. The infarct size following in-vivo I/R injury was not altered upon PD1-deficiency as determined by TTC staining. However, flow cytometry determined increased cell numbers of distinct leukocyte subsets during reperfusion-related inflammation. Conclusion Deficiency of PD1/PDL1 signalling shows distinct inflammatory changes in cardiac tissue at baseline and I/R injury. The results indicate that relevant PD1-related detrimental effects are not limited to complications from cancer therapy but can be expected in various forms of cardiovascular disease, hence requiring further investigations. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): Department of Cardiology and Vascular Medicine, West German Heart and Vascular Center, University Hospital Essen, Germany

Differential CD147 Functional Epitopes on Distinct Leukocyte Subsets.

CD147, a member of the immunoglobulin (Ig) superfamily, is widely expressed in several cell types. CD147 molecules have multiple cellular functions, such as migration, adhesion, invasion, energy metabolism and T cell activation. In particular, recent studies have demonstrated the potential application of CD147 as an effective therapeutic target for cancer, as well as autoimmune and inflammatory diseases. In this study, we elucidated the functional epitopes on CD147 extracellular domains in T cell regulation using specific monoclonal antibodies (mAbs). Upon T cell activation, the anti-CD147 domain 1 mAbs M6-1E9 and M6-1D4 and the anti-CD147 domain 2 mAb MEM-M6/6 significantly reduced surface expression of CD69 and CD25 and T cell proliferation. To investigate whether functional epitopes of CD147 are differentially expressed on distinct leukocyte subsets, PBMCs, monocyte-depleted PBMCs and purified T cells were activated in the presence of anti-CD147 mAbs. The mAb M6-1E9 inhibited T cell functions via activation of CD147 on monocytes with obligatory cell-cell contact. Engagement of the CD147 epitope by the M6-1E9 mAb downregulated CD80 and CD86 expression on monocytes and IL-2, TNF-α, IFN-γ and IL-17 production in T cells. In contrast, the mAb M6-1D4 inhibited T cell function via activation of CD147 on T cells by downregulating IL-2, TNF-α and IFN-γ. Herein, we demonstrated that certain epitopes of CD147, expressed on both monocytes and T cells, are involved in the regulation of T cell activation.

Neutrophil-specific deletion of Syk results in recruitment-independent stabilization of the barrier and a long-term improvement in cognitive function after traumatic injury to the developing brain

While traumatic brain injury (TBI) is the leading cause of death and disability in children, we have yet to identify those pathogenic events that determine the extent of recovery. Neutrophils are best known as “first responders” to sites of infection and trauma where they become fully activated, killing pathogens via proteases that are released during degranulation. However, this activational state may generate substantial toxicity in the young brain after TBI that is partially due to developmentally regulated inadequate antioxidant reserves. Neutrophil degranulation is triggered via a downstream signaling pathway that is dependent on spleen tyrosine kinase (Syk). To test the hypothesis that the activational state of neutrophils is a determinant of early pathogenesis and long-term recovery, we compared young, brain-injured conditional knockouts of Syk (sykf/fMRP8-cre+) to congenic littermates (sykf/f). Based upon flow cytometry, there was an extended recruitment of distinct leukocyte subsets, including Ly6G+/Ly6C− and Ly6G+/Ly6Cint, over the first several weeks post-injury which was similar between genotypes. Subsequent assessment of the acutely injured brain revealed a reduction in blood-brain barrier disruption to both high and low molecular weight dextrans and reactive oxygen species in sykf/fMRP8-cre+ mice compared to congenic littermates, and this was associated with greater preservation of claudin 5 and neuronal integrity, as determined by Western blot analyses. At adulthood, motor learning was less affected in brain-injured sykf/fMRP8-cre+ mice as compared to sykf/f mice. Performance in the Morris Water Maze revealed a robust improvement in hippocampal-dependent acquisition and short and long-term spatial memory retention in sykf/fMRP8-cre+ mice. Subsequent analyses of swim path lengths during hidden platform training and probe trials showed greater thigmotaxis in brain-injured sykf/f mice than sham sykf/f mice and injured sykf/fMRP8-cre+ mice. Our results establish the first mechanistic link between the activation state of neutrophils and long-term functional recovery after traumatic injury to the developing brain. These results also highlight Syk kinase as a novel therapeutic target that could be further developed for the brain-injured child.

The Role of Sinusoidal Endothelial Cells in the Axis of Inflammation and Cancer Within the Liver.

Liver sinusoidal endothelial cells (LSEC) form a unique barrier between the liver sinusoids and the underlying parenchyma, and thus play a crucial role in maintaining metabolic and immune homeostasis, as well as actively contributing to disease pathophysiology. Whilst their endocytic and scavenging function is integral for nutrient exchange and clearance of waste products, their capillarisation and dysfunction precedes fibrogenesis. Furthermore, their ability to promote immune tolerance and recruit distinct immunosuppressive leukocyte subsets can allow persistence of chronic viral infections and facilitate tumour development. In this review, we present the immunological and barrier functions of LSEC, along with their role in orchestrating fibrotic processes which precede tumourigenesis. We also summarise the role of LSEC in modulating the tumour microenvironment, and promoting development of a pre-metastatic niche, which can drive formation of secondary liver tumours. Finally, we summarise closely inter-linked disease pathways which collectively perpetuate pathogenesis, highlighting LSEC as novel targets for therapeutic intervention.

Exploring the relationships between amphibian (Xenopus laevis) myeloid cell subsets

The differentiation of distinct leukocyte subsets is governed by lineage-specific growth factors that elicit disparate expression of transcription factors and markers by the developing cell populations. For example, macrophages (Mφs) and granulocytes (Grns) arise from common granulocyte-macrophage progenitors in response to distinct myeloid growth factors. In turn, myelopoiesis of the Xenopus laevis anuran amphibian appears to be unique to other studied vertebrates in several respects while the functional differentiation of amphibian Mφs and Grns from their progenitor cells remains poorly understood. Notably, the expression of colony stimulating factor-1 receptor (CSF-1R) or CSF-3R on granulocyte-macrophage progenitors marks their commitment to Mφ- or Grn-lineages, respectively. CSF-1R is activated by the colony stimulating factor-1 (CSF-1) and interleukin (IL-34) cytokines, resulting in morphologically and functionally distinct Mφ cell types. Conversely, CSF-3R is ligated by CSF-3 in a process indispensable for granulopoiesis. Presently, we explore the relationships between X. laevis CSF-1-Mφs, IL-34-Mφs and CSF-3-Grns by examining their expression of key lineage-specific transcription factor and myeloid marker genes as well as their enzymology. Our findings suggest that while the CSF-1- and IL-34-Mφs share some commonalities, the IL-34-Mφs possess transcriptional patterns more akin to the CSF-3-Grns. IL-34-Mφs also possess robust expression of dendritic cell-associated transcription factors and surface marker genes, further underlining the difference between this cell type and the CSF-1-derived frog Mφ subset. Moreover, the three myeloid populations differ in their respective tartrate-resistant acid phosphatase, specific- and non-specific esterase activity. Together, this work grants new insights into the developmental relatedness of these three frog myeloid subsets.

Abstract PD5-12: Spatial mapping of the immune microenvironment in primary triple-negative breast cancer (TNBC) and association with neoadjuvant therapy response

Abstract Background: Emerging data suggest that some patients with TNBC could benefit from the addition of immune-based therapy. This observation is in part due to the significant association between distinct tumor-infiltrating lymphocytes (TILs) and prognosis in TNBC. Efforts to comprehensively characterize the immune microenvironment of TNBCs are critically important to gain a better understanding of the immune landscape and how it influences response to both standard chemotherapy and immunotherapy. We previously assessed both stromal TILs (sTILs) and intraepithelial TILs (iTILs) from pre-treatment tumor samples from patients enrolled on a phase II study of neoadjuvant gemcitabine, carboplatin and iniparib (PrECOG 0105; NCT00813956). We found that both iTILS and sTILS significantly associated with pathologic response. Furthermore, we assessed a novel 'in silico flow cytometry' gene expression-based method, CIBERSORT, designed to assess overall immune content and deconvolute the relative levels of distinct leukocyte subsets in tumors. Specific leukocyte subsets significantly associated with pCR included activated memory CD4+ T cells, CD8+ T cells and M1 macrophages (all p<0.05). To validate these findings and further explore spatial mapping of these immune cell subsets we undertook this analysis. Methods: We performed SPARTA - Spatial Perception And Regional Tumor Analysis - a multiplexed immunohistochemistry-based technology with data visualization and analysis modules that is designed to capture clinically relevant information about the tumor microenvironment in 72 pre-treatment FFPE tumor sections from patients enrolled on PrECOG 0105. SPARTA was used to test and analyze the coordinated expression of PD-L1, PD-1, CD45RO, CD4, CD8 and HLA-DRA in the study cohort from whole slide scanned images. Pathologic response at the time of surgery was evaluated using the residual cancer burden index. Germline BRCA1 and BRCA2 status was known for all patients. Results: Of 72 samples, 67 were evaluable for SPARTA analysis. Within a subset of tumors separately profiled by both SPARTA (FFPE/IHC microscopy) and CIBERSORT (Frozen/RNA GEP), we found significant correlation for clinically relevant TIL subpopulations, including for CD8+ T cells (r=0.83, p<0.0001), and activated memory CD4+ T cells (r=0.78, p<0.0001). On average, the SPARTA frequency of CD8, PD-L1 and CD4 expressing cell types were higher in samples from the pCR group when compared to the no pCR group. Subpopulation analysis revealed that, on average, the frequency of CD4+HLA-DRA+ cells were higher in samples from the pCR group when compared to the no pCR group. Samples from the pCR group were associated with a higher local spatial density of CD45RO+HLA-DRA+CD4+ cells when compared to the no pCR group. Location- and proximity-based analyses are ongoing and will be presented. Analyses based on germline BRCA1/2 mutant versus wild-type status, as well as associations between tumor mutation burden and neoantigen burden will also be presented. Conclusions: Spatial mapping of the immune microenvironment in primary TNBC reveals distinct immune cell populations associated with response to neoadjuvant platinum-based therapy. Citation Format: Telli ML, Vinayak S, Khododoust MS, Gruber JJ, Ford JM, Sanchez P, Banayan N, Azimi S, Tumeh PC, Newman AM, Alizadeh AA. Spatial mapping of the immune microenvironment in primary triple-negative breast cancer (TNBC) and association with neoadjuvant therapy response [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD5-12.

A monocyte gene expression signature in the early clinical course of Parkinson\u2019s disease

Microglia are the main immune cells of the brain and express a large genetic pattern of genes linked to Parkinson’s disease risk alleles. Monocytes like microglia are myeloid-lineage cells, raising the questions of the extent to which they share gene expression with microglia and whether they are already altered early in the clinical course of the disease. To decipher a monocytic gene expression signature in Parkinson’s disease, we performed RNA-seq and applied the two-sample Kolmogorov-Smirnov test to identify differentially expressed genes between controls and patients with Parkinson's disease and changes in gene expression variability and dysregulation. The gene expression profiles of normal human monocytes and microglia showed a plethora of differentially expressed genes. Additionally, we identified a distinct gene expression pattern of monocytes isolated from Parkinson’s disease patients at an early disease stage compared to controls using the Kolmogorov-Smirnov test. Differentially expressed genes included genes involved in immune activation such as HLA-DQB1, MYD88, REL, and TNF-α. Our data suggest that future studies of distinct leukocyte subsets are warranted to identify possible surrogate biomarkers and may lead to the identification of novel interventions early in the disease course.

Cryopreserved whole blood for the quantification of monocyte, T-cell and NK-cell subsets, and monocyte receptor expression by multi-color flow cytometry: Amethodological study based on participants from the canadian longitudinal study on aging.

Immunophenotyping by multi-color flow cytometry is arguably the best tool to identify and quantify distinct cell lineages from the peripheral blood and other biological fluids/tissues. Effective in both clinical and research settings, it can be used to estimate the frequency of a given cell type or measure its phenotypic or functional properties. Normally, immunophenotyping is performed in fresh or fractionated blood (i.e., PBMCs) the same day, or within 24 hours of collection; however, this may not be feasible for all study designs. We have previously shown that cryopreserved blood, a biospecimen that is simple and inexpensive to prepare, is comparable to fresh blood for the enumeration of major leukocyte cell types. For the following study, we sought to extend these observations to distinct subsets of: monocytes (classical, intermediate, and non-classical), T-cells (CD4/CD8 naïve, central and effector memory, senescent, and terminally differentiated, and regulatory T-cells), and NK-cells (CD56 bright and dim); we also examined the expression of monocyte cell-surface receptors CX3CR1, CCR2, TLR2, and TLR4. Our results indicate that cryopreserved blood is comparable to fresh blood; with exception to relatively rare subsets and lowly expressed receptors, the absolute or relative frequency of cell subsets generally correlated >0.80 between blood types, while monocyte receptor expressed was mostly >0.70. Furthermore, the day-to-day coefficient of variation for most cell subsets and parameters was below 20%. Given these findings, we suggest that cryopreserved peripheral blood be given greater consideration for studies in which the quantification of distinct leukocyte subsets is required. © 2018 International Society for Advancement of Cytometry.

Reduced Expression of Membrane Complement Regulatory Protein CD59 on Leukocytes following Lung Transplantation.

Cellular protection against undesired effects of complement activation is provided by expression of membrane-bound complement regulatory proteins including CD59. This protein prevents membrane attack complex formation and is considered to be involved in graft accommodation. Also, CD59 downregulates CD4+ and CD8+ T-cell activation and proliferation. It is unknown whether CD59 expression is affected by transplantation. The aim of this study was to evaluate the quantitative CD59 antigen expression on distinct leukocyte subsets following lung transplantation (n = 26) and to investigate whether this differs from pretransplantation (n = 9). The results show that CD59 expression on leukocytes is significantly lower posttransplantation compared with healthy controls (p = 0.002) and pretransplantation (p < 0.0001). Moreover, the CD59 expression diminishes posttransplantation on all distinct lymphocyte subsets (p < 0.02). This effect appeared to be specific for CD59 since the expression of other surface markers remained stable or inclined following transplantation. The highest antigen expression posttransplantation was observed on CD4+ T cells and monocytes (p ≤ 0.002). These findings show that CD59 expression on leukocytes diminishes posttransplantation, which could result in decreased resistance against complement and enhanced T-cell activation. If such reduction in CD59 expression also occurs on endothelial cells from the transplanted organ, this could lead to a change into a prothrombotic and proinflammatory phenotype.

Cortisol is More Important than Metanephrines in Driving Changes in Leukocyte Counts after Stroke

There are notable changes in the number of white blood cells (WBCs) after stroke, but the primary mediators of these changes are unclear. In this study, we assessed the role of the neuroendocrine and sympathetic nervous systems in stroke-induced changes of WBCs within distinct leukocyte subsets, as well as the effect of these changes on stroke outcomes. Patients were recruited within 72 hours after ischemic stroke; complete blood count with differential was obtained at set time points. The relationships among leukocyte numbers, cortisol, adrenocorticotropic hormone, interleukin-6, and metanephrines were assessed at 72 hours after stroke. Associations between abnormal leukocyte counts at 72 hours, poststroke infection, and 3-month outcomes were determined. A total of 114 subjects were enrolled. Severe stroke was associated with leukocytosis, neutrophilia, monocytosis, lymphopenia, and eosinopenia. At 72 hours after stroke, increased serum cortisol was independently associated with neutrophilia and lymphopenia. Abnormal leukocyte counts were not independently predictive of poststroke infection, but lymphopenia was associated with poor outcome (modified Rankin score >3) at 3 months after stroke (odds ratio = 22.86 [1.95, 267.65]; P = .01). Increased serum cortisol is independently associated with neutrophilia and lymphopenia after stroke. Lymphopenia is not an independent predictor of infections but is independently associated with worse outcome.

MicroRNA expression signatures in lungs of mice infected with Mycobacterium tuberculosis

Tuberculosis (TB) is a major public health concern worldwide; however the factors that account for resistance or susceptibility to disease are not completely understood. Although some studies suggest that the differential expression of miRNAs in peripheral blood of TB patients could be useful as biomarkers of active disease, their involvement during the inflammatory process in lungs of infected individuals is unknown. Here, we evaluated the global expression of miRNAs in the lungs of mice experimentally infected with Mycobacterium tuberculosis on 30 and 60 days post-infection. We observed that several miRNAs were differentially expressed compared to uninfected mice. Furthermore, we verified that the expression of miR-135b, miR-21, miR-155, miR-146a, and miR-146b was significantly altered in distinct leukocyte subsets isolated from lungs of infected mice, while genes potentially targeted by those miRNAs were associated with a diversity of immune related molecular pathways. Importantly, we validated the inhibition of Pellino 1 expression by miR-135b in vitro. Overall, this study contributes to the understanding of the dynamics of miRNA expression in lungs during experimental TB and adds further perspectives into the role of miRNAs on the regulation of immune processes such as leukocyte activation.

The Players: Cells Involved in Glomerular Disease.

Glomerular diseases are common and important. They can arise from systemic inflammatory or metabolic diseases that affect the kidney. Alternately, they are caused primarily by local glomerular abnormalities, including genetic diseases. Both intrinsic glomerular cells and leukocytes are critical to the healthy glomerulus and to glomerular dysregulation in disease. Mesangial cells, endothelial cells, podocytes, and parietal epithelial cells within the glomerulus all play unique and specialized roles. Although a specific disease often primarily affects a particular cell type, the close proximity, and interdependent functions and interactions between cells mean that even diseases affecting one cell type usually indirectly influence others. In addition to those cells intrinsic to the glomerulus, leukocytes patrol the glomerulus in health and mediate injury in disease. Distinct leukocyte types and subsets are present, with some being involved in different ways in an individual glomerular disease. Cells of the innate and adaptive immune systems are important, directing systemic immune and inflammatory responses, locally mediating injury, and potentially dampening inflammation and facilitating repair. The advent of new genetic and molecular techniques, and new disease models means that we better understand both the basic biology of the glomerulus and the pathogenesis of glomerular disease. This understanding should lead to better diagnostic techniques, biomarkers, and predictors of prognosis, disease severity, and relapse. With this knowledge comes the promise of better therapies in the future, directed toward halting pathways of injury and fibrosis, or interrupting the underlying pathophysiology of the individual diseases that lead to significant and progressive glomerular disease.

Abstract PR004: Tracking microbial sensing in colorectal cancer using bioorthogonal click chemistry

Abstract Just as the organs within an individual must communicate to function as a whole, the interactions of the microbiota with the host define local and systemic physiology. Within the intestine, pattern recognition receptors sense and respond to microbial products to promote tolerance or drive inflammation. In animal models and human patients with colorectal cancer, this innate immune sensing system fundamentally fails, leading to a highly inflammatory state and a tumorigenic environment. However, there is little known about the spatial and temporal distribution of the microbial products that signal through pattern recognition receptors within the tissue and how this relates to the immunological influence on tumor formation and progression. We present an interdisciplinary approach utilizing bioorthogonal click chemistry (i.e. reactions that can be performed in living organisms) to label and track components of commensal organisms. We have used this method to successfully label the capsular polysaccharide A (PSA) of the important human commensal, Bacteroides fragilis. Since PSA can signal through TLR-2 to stimulate regulatory T cells and protect mice against experimental colitis, this molecule serves as an excellent model to decipher commensal-host communication. After administration of fluorescently labeled B. fragilis to the murine host, we can trace PSA into specific host immune cells in the intestine to define the pathways by which protective or inflammatory responses develop. We are also using this approach to visualize the spatial and temporal changes in bacterial product acquisition within diseased tissue. In mouse models of colitis associated colorectal cancer, we observe differences in distinct leukocyte subsets associated with PSA in the gut-associated mesenteric lymph nodes. We are currently characterizing the role that these identified leukocytes may play in PSAs immunomodulatory activity and why this changes during colorectal cancer. We have also expanded on our chemical labeling approach by incorporating additional non-natural sugars and amino acids into the bacterial cell wall to fluorescently label lipopolysaccharide (LPS), capsular polysaccharide (CPS), and peptidoglycan (PGN). Employing this cutting-edge chemistry with modern methods in immunology and microscopy allows us to simultaneously track Toll-like receptor ligands (LPS, CPS) and NOD-like receptor ligands (PGN) from living commensal bacteria within host tissue. We are currently using this system to investigate whether certain cell types associate with distinct commensal molecules and how those pathways affect colorectal cancer progression. J.E.H. is supported by a Cancer Research Institute Irvington postdoctoral fellowship. Citation Format: Jason Hudak, Naama Geva-Zatorsky, Dennis L. Kasper. Tracking microbial sensing in colorectal cancer using bioorthogonal click chemistry. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr PR004.

The prognostic landscape of genes and infiltrating immune cells across human cancers.

Molecular profiles of tumors and tumor-associated cells hold great promise as biomarkers of clinical outcomes. However, existing data sets are fragmented and difficult to analyze systematically. Here we present a pan-cancer resource and meta-analysis of expression signatures from ∼18,000 human tumors with overall survival outcomes across 39 malignancies. By using this resource, we identified a forkhead box MI (FOXM1) regulatory network as a major predictor of adverse outcomes, and we found that expression of favorably prognostic genes, including KLRB1 (encoding CD161), largely reflect tumor-associated leukocytes. By applying CIBERSORT, a computational approach for inferring leukocyte representation in bulk tumor transcriptomes, we identified complex associations between 22 distinct leukocyte subsets and cancer survival. For example, tumor-associated neutrophil and plasma cell signatures emerged as significant but opposite predictors of survival for diverse solid tumors, including breast and lung adenocarcinomas. This resource and associated analytical tools (http://precog.stanford.edu) may help delineate prognostic genes and leukocyte subsets within and across cancers, shed light on the impact of tumor heterogeneity on cancer outcomes, and facilitate the discovery of biomarkers and therapeutic targets.

Abstract P5-04-03: Deconvoluting immune cell populations using ‘in silico flow cytometry’ with CIBERSORT: Association with neoadjuvant therapy response and genomic instability in TNBC

Abstract Background: Increased tumor infiltrating lymphocytes (TILs) are prognostic and predictive of therapy response in TNBC. CIBERSORT, a highly novel ‘in silico flow cytometry’ gene expression-based method, can assess the overall immune content and relative levels of distinct leukocyte subsets in tumors. Methods: We applied CIBERSORT to PrECOG 0105, a neoadjuvant trial of carboplatin, gemcitabine and iniparib for patients with clinical stage I-IIIA TN or BRCA1/2 mutation-associated BC. H&amp;E tumor sections from pre-therapy biopsies were evaluated for density of stromal (sTILs) and intratumoral (iTILs) lymphocytes. Pathologic response was assessed at definitive surgery. Overall immune content and fractions of 23 distinct leukocyte subsets were derived from Affymetrix U133 plus 2.0 arrays, using CIBERSORT. Using an input matrix of reference gene expression signatures of 23 purified leukocytes, this methodology can infer an unknown fraction of each cell type from tissue expression profiling, yielding a p value for deconvolution. All patients had BRCA1/2 genotyping and levels of tumor genomic instability were assessed by the homologous recombination deficiency (HRD) assay. Results: 57 pts had gene expression and TILs assessment. TILs, sTILs, and iTILs were significantly associated with a measure of absolute immune content determined by CIBERSORT, termed an ‘immune score’ (R 0.73, p&amp;lt;0.0001; R 0.70, p&amp;lt;0.0001; R 0.57, p&amp;lt;0.0001 respectively). Both iTILs and the CIBERSORT immune score were significantly associated with pathologic complete response (pCR; ypT0/is N0) in independent models. Specific leukocyte subsets significantly associated with pCR, included activated memory CD4 T cells, CD8 T cells, and M1 macrophages (all p&amp;lt;0.05). Regarding genomic instability measures, BRCA1/2 germline mutation status was not associated with TILs or immune score, whereas a high HRD assay score was significantly associated with the CIBERSORT immune score (p=0.038). Individual leukocyte subsets significantly associated with a high HRD assay score, included plasma cells, activated memory CD4 T cells, M1 macrophages, and unstimulated mast cells (all p&amp;lt;0.05). Conclusions: Neoadjuvant platinum-based therapy response significantly associates with both iTILs and CIBERSORT immune score. A measure of global genomic instability (HRD score) significantly associates with immune score alone. Enumeration of 23 leukocyte subsets in therapy-naïve TNBC by CIBERSORT revealed three distinct cell types that significantly predict pCR, two of which also associate with genomic instability. These results suggest an intimate interplay between genomic instability and immune infiltration, potentially shaping adaptive anti-tumor humoral immune responses, and thereby affecting neoadjuvant response in TNBC. Citation Format: Shaveta Vinayak, Aaron Newman, Sylvia Adams, Anosheh Afghahi, Kristin C Jensen, Sunil S Badve, James M Ford, Ash A Alizadeh, Melinda L Telli. Deconvoluting immune cell populations using ‘in silico flow cytometry’ with CIBERSORT: Association with neoadjuvant therapy response and genomic instability in TNBC [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-04-03.

Molecular Imaging of Inflammation in Atherosclerosis

Acute rupture of vulnerable plaques frequently leads to myocardial infarction and stroke. Within the last decades, several cellular and molecular players have been identified that promote atherosclerotic lesion formation, maturation and plaque rupture. It is now widely recognized that inflammation of the vessel wall and distinct leukocyte subsets are involved throughout all phases of atherosclerotic lesion development. The mechanisms that render a stable plaque unstable and prone to rupture, however, remain unknown and the identification of the vulnerable plaque remains a major challenge in cardiovascular medicine. Imaging technologies used in the clinic offer minimal information about the underlying biology and potential risk for rupture. New imaging technologies are therefore being developed, and in the preclinical setting have enabled new and dynamic insights into the vessel wall for a better understanding of this complex disease. Molecular imaging has the potential to track biological processes, such as the activity of cellular and molecular biomarkers in vivo and over time. Similarly, novel imaging technologies specifically detect effects of therapies that aim to stabilize vulnerable plaques and silence vascular inflammation. Here we will review the potential of established and new molecular imaging technologies in the setting of atherosclerosis, and discuss the cumbersome steps required for translating molecular imaging approaches into the clinic.