Cell Subset Markers Research Articles

An abnormal increase in CD26(-)CD28(-) cytotoxic effector CD4 and CD8 T cell populations in patients with systemic lupus erythematosus.

CD26 is a human T cell costimulatory molecule as well as a T cell subset marker, and increase of CD26+ T cells in inflamed tissues and peripheral blood has been reported in diverse autoimmune diseases. In contrast, our group has previously shown that levels of circulating CD26+ T cells are decreased in patients with systemic lupus erythematosus (SLE), although the role of reduced CD26 T cell surface expression in SLE pathology remains to be elucidated. In the present study, we conducted CD26-based T cell subset analyses utilizing peripheral blood mononuclear cells from 57 SLE patients and 31 healthy adult volunteers. We show that the increase in CD26(-) T cell population reflects the abnormal expansion of CD26(-)CD28(-) cytotoxic subsets of both CD8 T cells and CD4 T cells in SLE patients. Single cell RNA sequencing analysis of the CD26(-)CD28(-) CD4 and CD8 T cell populations reveals unique characteristics with similarities to natural killer T cells. In addition, the level of CD26(-)CD28(-) T cells is increased in some active stage SLE patients with renal manifestation. Meanwhile, effect of prednisolone treatment on these populations varies from patient to patient, with levels of these cytotoxic effector populations still being elevated in some inactive stage SLE patients. Taken together, our data suggest that analysis of these populations in SLE may be a useful tool to classify this markedly heterogeneous condition.

Single-cell Mayo Map (scMayoMap): an easy-to-use tool for cell type annotation in single-cell RNA-sequencing data analysis

BackgroundSingle-cell RNA-sequencing (scRNA-seq) has become a widely used tool for both basic and translational biomedical research. In scRNA-seq data analysis, cell type annotation is an essential but challenging step. In the past few years, several annotation tools have been developed. These methods require either labeled training/reference datasets, which are not always available, or a list of predefined cell subset markers, which are subject to biases. Thus, a user-friendly and precise annotation tool is still critically needed.ResultsWe curated a comprehensive cell marker database named scMayoMapDatabase and developed a companion R package scMayoMap, an easy-to-use single-cell annotation tool, to provide fast and accurate cell type annotation. The effectiveness of scMayoMap was demonstrated in 48 independent scRNA-seq datasets across different platforms and tissues. Additionally, the scMayoMapDatabase can be integrated with other tools and further improve their performance.ConclusionsscMayoMap and scMayoMapDatabase will help investigators to define the cell types in their scRNA-seq data in a streamlined and user-friendly way.

Simplified and Optimized Immune Score for Colorectal Cancer Microenvironment.

Immunoscore (IS) is an important evaluation method for the tumor immune microenvironment (TIME); however, formal IS analysis requires designated reagents and a specific digital pathology software and image data analysis. This study aimed to investigate whether simplified IS (s-IS) can substitute formal IS upon modifying the location of the assessment of the numbers of immune cells and verify that the addition of T cell subset markers to s-IS can enhance the prognostic impact in patients with colorectal cancer (CRC). A total of 82 CRC cases were included in this study. Immunohistochemical analysis was performed using CD3/CD8/CD45RO/FOXP3 on tissue specimens; the expression levels were calculated in the center and perimeter of the tumors using digital pathology. The clinical prognostic significance of the expression of these markers was investigated by concordance index comparison according to their location of assessment and combinations. In the univariate analysis, the CD3, CD8, and FOXP3 levels were significant prognostic factors. Moreover, for each T cell subset marker, the assessment of each T cell subset marker at the tumor perimeter had a stronger prognostic power than that in the tumor center. The modified s-IS (s-IS plus FOXP3 evaluation) was an independent prognostic factor for recurrence-free survival and overall survival through multivariate analysis and demonstrated the best prognostic power compared to other T subset marker combinations. In CRC, TIME evaluation could be simplified by assessing CD3- and CD8-positive T cells in the perimeter of the tumor, and additional FOXP3 evaluation would empower the ability of s-IS evaluation in prognostic assessment.

Infiltration of Mature KLRG1 Expressing Cytotoxic T Cells in Oral Lichen Planus.

Oral lichen planus (OLP) is a chronic inflammatory disease affecting oral mucosa. Its pathogenesis includes T cell infiltration. T cells may be naïve or in response to antigen stimulation, progress through differentiation stages. The differentiated states of T cells in OLP mucosa have not previously been reported. Available OLP microarray gene expression data from Gene Expression Omnibus were analyzed for markers of T cell cytotoxicity. Immunohistochemical studies of T cell subset markers CD4 and CD8 and the T cell differentiation marker killer cell lectin-like receptor G1 (KLRG1) were performed on paraffin embedded formalin fixed oral mucosa biopsy samples from 10 patients with OLP. Gene expression analysis of OLP oral mucosa samples disclosed increased transcript expression of KLRG1, CD8A, and granzyme K (GZMK). By immunohistochemistry, prominent CD4 + and CD8 + T cell infiltration was seen in all patient samples. KLRG1 + T cells were abundant, constituting a mean of 51% (range 40-65%) of the number of CD8 + T cells. KLRG1 + T cells localized at the epithelium and lamina propria junction, infiltrating both basal and intraepithelial regions and adjacent to both basal and intraepithelial keratinocytes. OLP oral mucosa T cell infiltration includes KLRG1 + highly differentiated cytotoxic T cells, suggesting continued antigen exposure driving T cells to a highly differentiated phenotype. The known phenotype of these cells, together with microarray detected increases in cytotoxic molecules, suggests that highly differentiated cytotoxic T cells contribute to oral mucosa injury in OLP.

P036 Identification and functional analysis of stromal subsets in experimental IBD mouse models

Abstract Background Single cell RNA sequencing data from the inflamed colon of inflammatory bowel disease (IBD) patients revealed the presence of different stromal cell/fibroblast subsets and showed their importance in IBD pathology. Up to now, it is unknown if and how the stroma cells affect disease progression. In this study we compared the stromal cell subsets in three IBD mouse models and explored the interaction between these stromal subsets and epithelial cells. Methods Stromal cell subset markers, including CD55, C-X-C motif chemokine 12 (CXCL12), podoplanin (PDPN), CD90 and CD73, were analyzed in the inflamed murine colon by flow cytometry in interleukin (IL)-10 knockout (KO) mice, dextran sulfate sodium(DSS)-induced colitis and the T cell transfer model. In order to explore the changes in stromal subsets upon colitis induction in vitro, short hairpin RNAs were used to silence target gene expression of these cellular markers in murine fibroblasts. Fibroblast proliferation and migration was studied in addition to the effects on immune cell trafficking and tissue regeneration. Results In all three colitis models, an increase in the total amount of stromal cells was observed. The percentages of PDPN+ and CD73+ stromal cells significantly increased during colitis in the IL-10 KO mice, while the percentage of CD55+, CD90+ and CXCL12+ stromal cells decreased. For the T cell transfer model, the abundance of CD73+, CD90+ and CD55+ stromal cells was enriched, while the percentage of CXCL12+ stromal cells was reduced. Interestingly, in contrast to the two other, in the DSS-mice the percentage of CXCL12+ stromal cells was significantly increased. In vitro experiments showed the importance of CD55 and CXCL12 for fibroblast proliferation and CD55 for their migration. Furthermore, the fibroblast supernatant obtained after the knockdown of CXCL12 decreased epithelial cell wound healing. On the contrary, knock-down of CD90 in the fibroblasts improved epithelial migration. Current experiments explore the immune regulatory properties of the fibroblast subsets. Conclusion The composition of stromal cell subsets is significantly changed in experimental colitis and differs between three established colitis mouse models. Therefore they may possibly reflect different human IBD subtypes observed in the clinic. Finally the defined subsets influence fibroblast behavior and epithelial regeneration.

Monitoring B cell alloresponses in rats

Antibody-mediated rejection is a major cause of graft failure in organ transplantation. For this reason, B cell responses are of particular interest to transplantation research. Rats are important model organisms for transplant studies, but B cell alloimmune assays and B cell subset markers are poorly established in rats. We alloimmunized rats by donor blood injection using the high responder rat strain combination Brown Norway (donor) and Lewis (recipient) rats. Using splenocytes from alloimmunized and control rats, we established assays to assess allospecific B cell proliferation and the capacity to generate allospecific B memory cells and alloantibody-secreting cells after antigenic rechallenge in vitro using a mixed lymphocyte reaction. Furthermore, we defined a simple gating and sorting strategy for pre- and post-germinal center follicular B cells, as well as non-switched and switched plasmablasts. Our protocols for assessing B cell alloresponses and B cell subsets in rats may help to accelerate research into the role of B cells and manipulation of humoral alloresponses in transplant research.

Application of an Activity-Based Probe to Determine Proteolytic Activity of Cell Surface Cathepsin G by Mass Cytometry Data Acquisition.

During an immune response, cathepsin G (CatG) takes on the role of adaptive and innate immunity and the outcome depends on the localization of CatG. Soluble, cell surface-bound, or intracellular CatG is also responsible for pathophysiology conditions. We applied the activity-based probe MARS116-Bt to mass cytometry by time-of-flight to analyze CatG activity on the cell surface of immune cells. The phosphonate warhead of MARS116-Bt binds covalently to the serine amino acid residue S195 of the catalytic center and thereby CatG activity can be detected. This method contributes to observing the activation or inhibition status of cells during pathogenesis of diseases and enables accurate data acquisition from complex biological samples with a vast panel of cell subset markers in a single-cell resolution.

Dissecting the immune landscape of tumor draining lymph nodes in melanoma with high-plex spatially resolved protein detection.

In melanoma patients, microscopic tumor in the sentinel lymph-node biopsy (SLN) increases the risk of distant metastases, but the transition from tumor in the SLN to metastatic disease remains poorly understood. Fluorescent staining for CD3, CD20, CD11c, and DNA was performed on SLN tissue and matching primary tumors. Regions of interest (ROI) were then chosen geometrically (e.g., tumor) or by fluorescent cell subset markers (e.g., CD11c). Each ROI was further analyzed using NanoString Digital Spatial Profiling high-resolution multiplex profiling. Digital counts for 59-panel immune-related proteins were collected and normalized to account for system variation and ROI area. Tumor regions of SLNs had variable infiltration of CD3 cells among patients. The patient with overall survival (OS) > 8years had the most CD11c- and CD3-expressing cells infiltrating the SLN tumor region. All patients had CD11c (dendritic cell, DC) infiltration into the SLN tumor region. Selecting ROI by specific cell subtype, we compared protein expression of CD11c cells between tumor and non-tumor/normal tissue SLN regions. Known markers of DC activation such as CD86, HLA-DR, and OX40L were lowest on CD11c cells within SLN tumor for the patient with OS < 1year and highest on the patient with OS > 8years. We demonstrate the feasibility of profiling the protein expression of CD11c cells within the SLN tumor. Identifying early regulators of melanoma control when the disease is microscopically detected in the SLN is beneficial and requires follow-up studies in a larger cohort of patients.

In vivo liposomal delivery of PPARα/γ dual agonist tesaglitazar in a model of obesity enriches macrophage targeting and limits liver and kidney drug effects.

Macrophages are important regulators of obesity-associated inflammation and PPARα and -γ agonism in macrophages has anti-inflammatory effects. In this study, we tested the efficacy with which liposomal delivery could target the PPARα/γ dual agonist tesaglitazar to macrophages while reducing drug action in common sites of drug toxicity: the liver and kidney, and whether tesaglitazar had anti-inflammatory effects in an in vivo model of obesity-associated dysmetabolism.Methods: Male leptin-deficient (ob/ob) mice were administered tesaglitazar or vehicle for one week in a standard oral formulation or encapsulated in liposomes. Following the end of treatment, circulating metabolic parameters were measured and pro-inflammatory adipose tissue macrophage populations were quantified by flow cytometry. Cellular uptake of liposomes in tissues was assessed using immunofluorescence and a broad panel of cell subset markers by flow cytometry. Finally, PPARα/γ gene target expression levels in the liver, kidney, and sorted macrophages were quantified to determine levels of drug targeting to and drug action in these tissues and cells.Results: Administration of a standard oral formulation of tesaglitazar effectively treated symptoms of obesity-associated dysmetabolism and reduced the number of pro-inflammatory adipose tissue macrophages. Macrophages are the major cell type that took up liposomes with many other immune and stromal cell types taking up liposomes to a lesser extent. Liposome delivery of tesaglitazar did not have effects on inflammatory macrophages nor did it improve metabolic parameters to the extent of a standard oral formulation. Liposomal delivery did, however, attenuate effects on liver weight and liver and kidney expression of PPARα and -γ gene targets compared to oral delivery.Conclusions: These findings reveal for the first time that tesaglitazar has anti-inflammatory effects on adipose tissue macrophage populations in vivo. These data also suggest that while nanoparticle delivery reduced off-target effects, yet the lack of tesaglitazar actions in non-targeted cells such (as hepatocytes and adipocytes) and the uptake of drug-loaded liposomes in many other cell types, albeit to a lesser extent, may have impacted overall therapeutic efficacy. This fulsome analysis of cellular uptake of tesaglitazar-loaded liposomes provides important lessons for future studies of liposome drug delivery.

Abstract 4560: A cost-effective and rapid assay for profiling of immunobiology and the development of predictive signatures for response to immunotherapies

Abstract Introduction: Gene expression profiling is an important tool to monitor the human immune response to different antitumor agents for drug development and to identify predictive signatures for response to immunotherapies. We have developed and analytically validated a gene expression assay panel, namely the AdvantaTM Immuno-Oncology Gene Expression Assay (AdvantaTM I/O GE Assay), on the Fluidigm BiomarkTM HD system, a microfluidic real-time PCR system, for investigation of antitumor agent effects on immune inhibitory pathways. The assay panel consists of 170 unique gene targets in categories such as T cell subset markers (e.g., CD4), cytokines and chemokines (e.g., IFNg), immune regulation (e.g., PD-L1, PD-L2) and immune cell-fate markers (e.g., NK) associated with immune-checkpoint inhibitory pathways for cancer treatments. Method: The Assay consists of Fluidigm Dynamic Array™ IFCs, an assay panel and RT-PCR reagents. The assay panel is composed of TaqMan™ gene expression assays provided in two 96-well plates: 96 assays on one and 79 assays on another with 17 unused assay wells available for customization. Each plate contains five housekeeping genes for assay normalization. This assay configuration allows customers to test 96 samples on two Fluidigm 96.96 Dynamic Array™ IFCs. Two primer pools for preamplification, and a positive control have been developed along with a 24.192 Dynamic Array™ IFC which enables 24 samples and the complete panel, 170 genes to be run on a single IFC. The assay has been analytically validated by a third party (Q2/EA) on the 96.96 IFC. This study is to verify the assay and kit reagents on the 24.192 IFC. A universal RNA was reversely transcribed into cDNA and pre-amplified with the preamp primer pools for all 170 genes. Three tissue cDNAs (lymph node, brain and Jurkat cells) and the positive control, which contains all 170 targets, were used to evaluate amplification efficiency, linearity and reproducibility. Each sample was tested in three replicates on 24.192 IFCs in six independent runs. Results: The results show an average amplification efficiency across all assays of 98.2% and linearity (RSQ) 0.999 for universal RNA. The efficiency and linearity for the positive control are 0.999 and 0.998, respectively. The within-run replicate correlation (RSQ) is 0.997 for the universal RNA, 0.992 for Jurkat cDNA, 0.986 for brain cDNA, and 0.990 for lymph node cDNA. The between-IFC replicate correlation (RSQ) is 0.981 for universal RNA, 0.982 for Jurkat cDNA, 0.972 for brain cDNA and 0.977 for lymph node cDNA. Gene expression signatures for individual tissues were observed. Conclusions: The AdvantaTM I/O GE Assay on 24.192 IFC and the Fluidigm BiomarkTM HD system provides high amplification efficiency, excellent linearity and reproducibility and reduces the cost of routine laboratory testing for both immune checkpoint research and drug development. Citation Format: Peilin Chen, Sandy Spurgeon, Jing Wang, Michael Gonzales, Lianne McLean, Tom Goralski. A cost-effective and rapid assay for profiling of immunobiology and the development of predictive signatures for response to immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4560.

Abstract 2092: Tumor-infiltrating myeloid cells: Using MultiOmyx to distinguish between TAMs, TANs, and MDSCs in the pancreatic tumor microenvironment

Abstract Myeloid cells are the primary recruited effector cells during inflammation. A subset of these, consisting primarily of tumor-associated M1/M2 macrophages (TAMs), tumor-associated neutrophils (TANs), and myeloid-derived suppressor cells (MDSCs), accumulate in tumors where they establish an inflammatory tumor microenvironment (TME) that is favorable for tumor progression (1-3). While these tumor-infiltrating myeloid cells (TIMs) are known to be major regulators of tumor-associated immune-suppression, angiogenesis, metastasis, and resistance to anti-cancer therapy much still remains to be understood about how they function within the TME and how they co-operate with tumor-resident lymphocytes to regulate antitumor immunity. Targeting TIMs to either eliminate them or to convert them from their immune-suppressive to an immune-stimulatory state has emerged as a new strategy complementing current cancer immunotherapy strategies. However, a major impediment to understanding the complexity of the distinct functions of subsets of TIMs and their spatial distribution within the TME is the ability to phenotypically characterize TIMs in FFPE tissues by standard immunohistochemistry, as most IHC studies do not utilize a panel of antibodies broad enough to characterize multiple myeloid cell subsets in the same sample. We will be using MultiOmyx, a proprietary, multiplexing assay with similar staining characteristics as standard IHC stains but with the significant advantage that up to 60 protein biomarkers can be interrogated from a single FFPE section (4). MultiOmyx protein immunofluorescence (IF) assays utilize a pair of directly conjugated Cyanine dye-labeled (Cy3, Cy5) antibodies per round of staining. Each round of staining is imaged and followed by novel dye inactivation chemistry, enabling repeated rounds of staining and deactivation. Using this MultiOmyx multiplexing assay in combination with a proprietary algorithm that takes into account the staining pattern for each specific biomarker, we will seek to identify different subsets of TIMs and their spatial relationship in tumor tissue from patients with pancreatic ductal adenocarcinoma (PDAC), characterized by an excessive amount of desmoplastic stroma seeded with inflammatory cells. TIM Panel: CD11b, CD14, CD15, CD16, CD33, CD68, CD163, HLA-DR, Arginase, PANCK. Human Myeloid Cell Subset Markers: M1 TAMs (CD68+HLA-DR+CD163-), M2 TAMs (CD68+CD163+HLA-DR+), TANs (CD11b+CD15+CD16+HLA-DR+Arginase+), M-MDSCs (CD11b+CD14+CD33+HLA-DRLoCD15-), G-MDSC (CD11b+CD15+CD33+Arginase+HLA-DRLoCD14-).

Spatial rearrangement of Purkinje cell subsets forms the transverse and longitudinal compartmentalization in the mouse embryonic cerebellum

AbstractTransversely oriented lobules and longitudinally arrayed stripes of Purkinje cell subsets subdivide the cerebellar cortex into multiple compartments that are involved in diverse functions. In the mammalian cerebellum, anterior, and posterior lobules, which are involved in somatosensorimotor function, show an alternation of aldolase C (zebrin II) ‐positive and ‐negative stripes, whereas the central lobules (lobules VIb–VII and crus I), which are implicated in nonmotor functions, show a laterally expanded arrangement solely of aldolase C‐positive stripes. To understand the developmental process of this compartmental pattern, we identified groups of Purkinje cell subsets in the entire mouse cerebellum at embryonic day (E) 14.5 by staining Purkinje cell subset markers. We then tracked four major domains of Protocadherin 10 (Pcdh10)‐positive Purkinje cell subsets (medial, dorsal, central, and mid‐lateral subsets), which were clearly demarcated during E14.5–17.5. These domains of Purkinje cell subsets shifted predominantly in the longitudinal direction to be positioned in the anterior and posterior lobules. However, a particular portion of the medial and mid‐lateral domains, and the whole of the central domain shift in the lateral direction to be positioned in the central lobules. The results indicate that while the longitudinal shift of domains of Purkinje cell subsets forms the longitudinally striped compartments in the anterior and posterior cerebellum, the lateral shift of particular domains of Purkinje cell subsets underlies the laterally expanded arrangement of stripes in central lobules. Thus, the rearrangement of Purkinje cell subsets in the embryonic cerebellum is critically related to the compartmental organization in the mammalian cerebellum.

Spatial rearrangement of Purkinje cell subsets forms the transverse and longitudinal compartmentalization in the mouse embryonic cerebellum.

Transversely oriented lobules and longitudinally arrayed stripes of Purkinje cell subsets subdivide the cerebellar cortex into multiple compartments that are involved in diverse functions. In the mammalian cerebellum, anterior, and posterior lobules, which are involved in somatosensorimotor function, show an alternation of aldolase C (zebrin II) -positive and -negative stripes, whereas the central lobules (lobules VIb-VII and crus I), which are implicated in nonmotor functions, show a laterally expanded arrangement solely of aldolase C-positive stripes. To understand the developmental process of this compartmental pattern, we identified groups of Purkinje cell subsets in the entire mouse cerebellum at embryonic day (E) 14.5 by staining Purkinje cell subset markers. We then tracked four major domains of Protocadherin 10 (Pcdh10)-positive Purkinje cell subsets (medial, dorsal, central, and mid-lateral subsets), which were clearly demarcated during E14.5-17.5. These domains of Purkinje cell subsets shifted predominantly in the longitudinal direction to be positioned in the anterior and posterior lobules. However, a particular portion of the medial and mid-lateral domains, and the whole of the central domain shift in the lateral direction to be positioned in the central lobules. The results indicate that while the longitudinal shift of domains of Purkinje cell subsets forms the longitudinally striped compartments in the anterior and posterior cerebellum, the lateral shift of particular domains of Purkinje cell subsets underlies the laterally expanded arrangement of stripes in central lobules. Thus, the rearrangement of Purkinje cell subsets in the embryonic cerebellum is critically related to the compartmental organization in the mammalian cerebellum.

Aging effects on T-bet expression in human B cell subsets

In order to compare human and mouse B cell subset markers, we evaluated T-bet expression in human B cell subsets from individuals of different ages. We found T-bet expressed in unstimulated memory more than naïve B cells, and more in young individuals. TLR7 stimulation up-regulated T-bet in all B cell subsets from young and elderly individuals, and more in the elderly. By fold-increase the best effect was seen in subsets of the elderly and especially in those that undergo class switch (naïve and IgM). We also evaluated CD11c expression, as T-bet+CD11c+ B cells are expanded in healthy elderly individuals and also in patients with autoimmunity. Similar to T-bet, CD11c expression was higher in memory than in naïve B cells, but no differences were observed between young and elderly individuals. After TLR7 stimulation, CD11c increases in all B cell subsets (especially in naïve and IgM) from the elderly.

Clinical significances of the CD14brightCD16bright cell subset in peripheral blood of patients with gastric cancer

Objective To study the clinical significances of CD14brightCD16bright cell subset in peripheral blood of patients with gastric cancer (GC). Methods The CD14brightCD16bright cells in peripheral blood samples collected from 124 patients with gastric cancer (GC), 130 patients with chronic gastritis (CG) and 80 normal healthy controls (HC) were measured by using flow cytometry. Differences in the CD14brightCD16bright cells between different groups were analyzed with the Mann-Whitney U test. The feasibility of using CD14brightCD16bright cells as a potential biomarker for differentiating GC patients from CG was assessed by using the receiver operating characteristic (ROC) curve analysis. Correlations between the CD14brightCD16bright cells and clinicopathologic parameters of GC were analyzed with multivariate correlation analysis. Results The percentages of CD14brightCD16bright cells in peripheral blood samples and in CD14bright monomuclear cells collected from the patients with GC [median: 0.38% (0.23%-0.52%) and 6.61% (4.23%-9.56%)] were significantly higher than those of the CG and HC groups [median: 0.11% (0.07%-0.15%) and 5.08% (3.35%-6.42%); median: 0.05% (0.03%-0.07%) and 5.09% (4.20%-7.40%)] (P<0.01). The area under the ROC curve for CD14brightCD16bright cells in the peripheral blood was 0.934 (95%CI: 0.900-0.968) indicating that the value of CD14brightCD16bright cells in the diagnosis of GC was much higher than that of alpha fetoprotein (AFP), cacino-embryonic antigen (CEA) and carbohydrate antigen CA199. The area under the ROC curve for combined multi-markers by using logistic model (CD14brightCD16bright cell subset and serum tumor markers) was 0.947 (95%CI: 0.920-0.973). The CD14brightCD16bright cells were closely associated with lymphocyte cells (P<0.01). Conclusion The CD14brightCD16bright cells were dramatically increased in the peripheral blood of patients with gastric cancer, which could be used as a biomarker in the diagnosis of gastric cancer. Key words: Mononuclear cell; Gastric cancer; CD14brightCD16bright cell subset

Peripheral blood-derived virus-specific memory stem T cells mature to functional effector memory subsets with self-renewal potency.

Memory T cells expressing stem cell-like properties have been described recently. The capacity of self-renewal and differentiation into various memory/effector subsets make them attractive for adoptive T cell therapy to combat severe virus infections and tumors. The very few reports on human memory stem T cells (T(SCM)) are restricted to analyses on polyclonal T cells, but extensive data on Ag-specific T(SCM )are missing. This might be due to their very low frequency limiting their enrichment and characterization. In this article, we provide functional and phenotypic data on human viral-specific T(SCM), defined as CD8(+)CD45RA(+)CCR7(+)CD127(+)CD95(+). Whereas <1% of total T cells express the T(SCM) phenotype, human CMV-specific T(SCM) can be detected at frequencies similar to those seen in other subsets, resulting in ∼ 1 /10,000 human CMV-specific T(SCM). A new virus-specific expansion protocol of sort-purified T(SCM) reveals both upregulation of various T cell subset markers and preservation of their stem cell phenotype in a significant proportion, indicating both self-renewal and differentiation potency of virus-specific T cells sharing their TCR repertoire. Furthermore, we describe a simplified culture protocol that allows fast expansion of virus-specific T(SCM) starting from a mixed naive T/T(SCM) pool of PBLs. Due to the clinical-grade compatibility, this might be the basis for novel cell therapeutic options in life-threatening courses of viral and tumor disease.

Utilizing dual-costimulation through CD134 and CD137 with varying levels of antigen presentation to elicit heterogeneous CD4+ T cell responses (IRC9P.703)

Abstract Many cancer vaccines target CD8 T cells, but cytotoxic CD4+ T cells are emerging as important players. Our lab utilized a dual-costimulation immunotherapy approach through CD134 (OX40) and CD127 (4-1BB) agonist antibodies together with T cells specific to a widespread antigen which induced cytotoxic CD4+ T cell differentiation. Drawing upon a novel transgenic mouse model where CD11c+ cells present a specific antigen, we now demonstrate that antigen-specific dual-costimulated CD4+ T cells can be heterogeneous with differential expression of Eomes, KLRG1, GzmB, and the IL-33R (ST2). Specifically, in one transgenic mouse line with lower levels of CD11c antigen presentation, CD4+ T cells developed into Eomes+KLRG1+GzmB- cells. Despite comparable costimulation however, CD4+ T cells developed into Eomes-KLRG1-GzmB+ cells in a mouse line with greater levels of CD11c antigen presentation. Analysis of known helper T cell transcription factors and cytokines also showed expression of other helper T cell subset markers in these cells. Further, the capacity of these cells to become activated and kill target cells is currently being investigated with an in vitro assay. These findings are novel, demonstrating that cytotoxic CD4+ T cells may in fact be a heterogeneous population influenced by antigen presentation levels. Thus, this data should greatly impact immunotherapeutic targeting of cytotoxic CD4+ T cells.

Identification of novel dendritic cell subset markers in human blood

Human dendritic cells (DC) are key regulators of innate and adaptive immunity that can be divided in at least three major subpopulations: plasmacytoid DC (pDC), myeloid type 1 DC (mDC1) and myeloid type 2 DC (mDC2) exhibiting different functions. However, research, diagnostic and cell therapeutic studies on human DC subsets are limited because only few DC subset markers have been identified so far. Especially mDC2 representing the rarest blood DC subset are difficult to be separated from mDC1 and pDC due to a paucity of mDC2 markers. We have combined multiparameter flow cytometry analysis of human blood DC subsets with systematic expression analysis of 332 surface antigens in magnetic bead-enriched blood DC samples. The initial analysis revealed eight novel putative DC subset markers CD26, CD85a, CD109, CD172a, CD200, CD200R, CD275 and CD301 that were subsequently tested in bulk peripheral blood mononuclear cell (PBMC) samples from healthy blood donors. Secondary analysis of PBMC samples confirmed three novel DC subset markers CD26 (dipeptidyl peptidase IV), CD85a (Leukocyte immunoglobulin-like receptor B3) and CD275 (inducible costimulator ligand). CD85a is specifically expressed in mDC1 and CD26 and CD275 represent novel mDC2 markers. These markers will facilitate human DC subset discrimination and additionally provide insight into potentially novel DC subset-specific functions.

Glioma Grade Is Associated with the Accumulation and Activity of Cells Bearing M2 Monocyte Markers

This study is directed at identifying the cell source(s) of immunomodulatory cytokines in high-grade gliomas and establishing whether the analysis of associated markers has implications for tumor grading. Glioma specimens classified as WHO grade II-IV by histopathology were assessed by gene expression analysis and immunohistochemistry to identify the cells producing interleukin (IL)-10, which was confirmed by flow cytometry and factor secretion in culture. Finally, principal component analysis (PCA) and mixture discriminant analysis (MDA) were used to investigate associations between expressed genes and glioma grade. The principle source of glioma-associated IL-10 is a cell type that bears phenotype markers consistent with M2 monocytes but does not express all M2-associated genes. Measures of expression of the M2 cell markers CD14, CD68, CD163, and CD204, which are elevated in high-grade gliomas, and the neutrophil/myeloid-derived suppressor cell (MDSC) subset marker CD15, which is reduced, provide the best index of glioma grade. Grade II and IV astrocytomas can be clearly differentiated on the basis of the expression of certain M2 markers in tumor tissues, whereas grade III astrocytomas exhibit a range of expression between the lower and higher grade specimens. The content of CD163(+) cells distinguishes grade III astrocytoma subsets with different prognosis.

Differential Changes in Expression of Intestinal Antimicrobial Peptide Genes During Ascaris lumbricoides Infection in Zambian Adults Do Not Respond to Helminth Eradication

Background. Intestinal helminthiasis modulates immune responses to vaccines and environmental allergens. To explore the impact on intestinal host defense, we assessed expression of antimicrobial peptide genes, together with T cell subset markers and cytokines, in patients with ascariasis before and after treatment.Methods. Case patients (n = 27) and control subjects (n = 44) underwent enteroscopy for collection of jejunal biopsy specimens, which were used in quantitative, real-time reverse-transcription polymerase chain reaction for a range of host defense genes; blood samples were also analyzed simultaneously.Results. The level of gene expression (mRNA) of HD5, hBD1, and LL-37 was lower in case patients than in control subjects, and the level of expression of HD6 was increased. However, after successful eradication, there was no trend to values seen in control subjects. Helminthiasis was associated with increased intestinal expression of the Th1 genes T-bet and interferon-γ. In peripheral blood mononuclear cells (PBMCs), a mixed profile of T cell markers and cytokines was increased. Ascaris-induced down-regulation of HD5 was observed in individuals with higher RORγt expression in PBMCs, but we found no evidence that this was mediated by circulating interleukin-22.Conclusions. Human ascariasis was associated with changes in antimicrobial peptide gene expression and immunological markers. Such changes may have implications for susceptibility to infectious disease and responsiveness to oral vaccines in tropical populations.