Optimizing Assays to Find Rare Antigen-Specific T cells in Cryopreserved PBMCs

Immunomonitoring of T cell based immune responses spans a wide range of therapeutic applications such as infectious and autoimmune diseases and is particularly important for vaccine research. Regardless of the therapeutic application, immunomonitoring can be a daunting task due to the variability of methods and protocols available. There are several commonly used functional assays for the enumeration of antigen specific CD8+ T cells and there is great variability in the protocols that are used for these assays. Thus, making it increasing difficult to thoroughly interpret data obtained from multi-center clinical trials and to compare results between laboratories. In order to address some of the issues associated with immunomonitoring of clinical trials, the Association for Immunotherapy of Cancer (CIMT) formed a CIMT monitoring panel tasked to standardize protocols for assaying T cell antigen immune responses. Thirteen centers from 6 different European countries participated in this study. They were given the same samples and asked to determine the number of antigen specific T cells and assess their antigen specific function using tetramer staining and a functional assay of their choice. Common techniques used for monitoring antigen induced immune responses included ELISPOT assays, HLA-multimer staining and intracellular cytokine staining (ICS).

Pre-tested samples of peripheral blood mononuclear cells (PBMC), synthetic peptides, and PE-conjugated HLA-tetramers were distributed to each center. Using HLA-typed healthy volunteers, PBMCs were isolated by Ficoll density gradient separation. Each sample was tested for T cell reactivity against CMV and influenza. All centers received an HLA-A negative control as well as HLA-A positive samples consisting of a combination of CMV and influenza reactive PBMCs. The study comprised of 2 phases; Phase I consisted of all centers performing the assays with their commonly used protocols, and in Phase II each center received optimized protocols based on the findings from Phase I.

For Phase I’s tetramer-staining assay, the laboratories could choose to stain samples with antibodies (Ab) for CD8+ alone, CD3+CD8+, or CD4+ CD8+ and use their preferred Ab clone, fluorescent dye, and Ab concentration. For the functional assays synthetic peptides were provided and each group could choose either the INF-γ ELISPOT assay, FACS-based intracellular INF-γ staining or both with their antigen concentration of choice ranging from 1-10 g/ml. To reduce variability in FACS analysis, sample plots were provided as well as gate settings and quadrants. Tetramer-staining data reported included; number of viable cells post-thawing, cytometer model, number of lymphocytes and/or CD8+ cells analyzed. Data was presented as percent of tetramer-positive cells among CD8+, CD3+CD8+, or CD4+ lymphocytes depending on what antibody cocktail was chosen. For the functional assays each center reported the type of ELISPOT plates used, reagents and conditions used, and number cells tested.

Tetramer results from the Phase I study showed the number CD8+ cells analyzed significantly affected the sensitivity of tetramer staining. Antigen-specific T cell reactivity when less than 30,000 CD8+ T cells were counted resulted in only 70% responsiveness detected. In contrast, when more than 30,000 CD8+ cells were counted, an 89% response was observed. Although, when antigen-specific T cells were present at high frequencies the number of counted cells did not matter. Interestingly, Ab clone variability, Ab concentration, or cytometer type did not result in any significant differences. Thus, the main factors affecting antigen-specific T cell reactivity by tetramer staining is the number of CD8+ cells used. For Phase II it was then recommended at least 1 x106 PBMCs are used for this assay.

The majority of groups chose the INF-γ ELISPOTas their functional assay. Results showed a large amount of heterogeneity between the centers. Some centers included a resting phase after thawing the cells, of 2-20 hours, resulting in 73% positive reactivity (number of spot forming cells per seeded PBMC). In contrast, not allowing a resting phase resulted in only detecting 30% of the positive cells. Additionally, intra-center replicate reproducibility was significantly affected by the number of replicates used, where duplicates often failed the Student t test and triplicates were sufficient to reach statistical significance. Addition of allogenic-APCs for binging and presentation of the synthetic peptides was found to have a negative effect on detection response (28% of all responses vs. 58%). When looking at the number of cells seeded per well, those with more than 4 x105 PBMC detected 71% positive samples and those with less than 4 x105 only detected 43%. Granted, when antigen specific T cells were available at high frequencies the number of counted cells did not affect the response rates. Consequently, Phase II’s minimum requirements for the INF-γ ELISPOT protocol included: (1) triplicates should be performed for each test antigen (2) avoid using allogenic-APCs (3) include a resting phase (4) use over 4 x 105 PBMCs per well.

Another interesting finding from this study was that lab experience in performing these assays had no effect on the performance of the assays compared to labs that had just adopted the techniques. Further highlighting the importance of developing standardized protocols for immunomonitoring assays. This study did not however, address specific detection limits for the ELISPOT assays, the variability between ELISPOT plate readers, nor serum source effects on background and specificity. In addition, it was not reported whether live/dead cell stains where included in the tetramer assays and how combinations of these may have had an effect on the sensitivity of the assay.

Overall, this study identified several factors that should be generally implemented when performing tetramer staining and INF-γ ELISPOT assays with cryopreserved PBMC samples. Furthermore, these protocol modifications are particularly important when assaying antigen-specific T cell populations present at low frequencies.


The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays. Britten CM, Gouttefangeas C, Welters MJ, Pawelec G, Koch S, Ottensmeier C, Mander A, Walter S, Paschen A, Müller-Berghaus J, Haas I, Mackensen A, Køllgaard T, thor Straten P, Schmitt M, Giannopoulos K, Maier R, Veelken H, Bertinetti C, Konur A, Huber C, Stevanović S, Wölfel T, van der Burg SH. Cancer Immunol Immunother. 2008 Mar;57(3):289-302. Epub 2007 Aug 25.

A theory of everything: commensal gut bacteria link environmental exposures to sex hormones in modulating autoimmunity

Whether an individual develops autoimmunity depends on how environmental and genetic factors interact to influence immune function. Genome wide association studies have revealed numerous risk alleles associated with diverse autoimmune diseases and small animal models have helped parse the effects of some of these gene variants on specific components of the immune system (see previous post). These approaches, however, largely ignore two of the more perplexing aspects of autoimmunity: (1) the strong female predominance of many autoimmune diseases and (2) the high incidence of autoimmunity in industrialized areas of the world compared to poorer, rural areas. The issue of sexual dimorphism in autoimmunity has led investigators to surmise that sex hormones are potent modulators of immune function. Indeed, altering estrogen, progesterone, or testosterone levels can influence the progression of autoimmune reactions in animals and people, although the specific mechanisms underpinning these phenomena remain controversial1. The increasing incidence of autoimmunity in the industrialized world has given rise to the “hygiene hypothesis”: exposure to specific microbes early in life is necessary for a fully-functioning immune system. These beneficial exposures are lacking in modernized, clean cities which contributes to immunological derangement and self-reactivity. Evidence supporting the hygiene hypothesis has accumulated in recent years to the point that clinical trials are in progress testing whether purposeful infestation with parasitic worms (thought to be the major microbial exposure missing from a modern upbringing) can control inflammatory bowel disease and multiple sclerosis2.


In a recent paper in Science Magazine, Dr. Jayne Danska’s group at the University of Toronto present an intriguing hypothesis that unifies the roles of early-life microbial exposure and sex hormone levels in driving an autoimmune response3. The group, with lead author Dr. Janet Markle, used a well-established mouse model of type-1 diabetes (T1D)—the non-obese diabetic (NOD) mouse—that is genetically predisposed to develop spontaneous, immune-mediated destruction of beta-islets at around 15 weeks of age, causing diabetes. Similar to several human autoimmune diseases, NOD mice have a 2:1 female-to-male sex bias in progressing to diabetes. In agreement with this, Markle et al. found that male mice housed under standard laboratory conditions were protected from developing diabetes compared to female mice. However, if NOD mice were born and raised in germ-free conditions (i.e. their intestines were never colonized by commensal bacteria) males developed the disease at the same rate as females. In addition, the environment in which the mice were raised impacted their levels of sex hormones: male mice raised in germ-free conditions had lower serum testosterone levels than those raised in standard conditions while germ free female mice had higher testosterone levels than those kept in standard cages. These data supported the conclusion that colonization with commensal bacteria was responsible for the protection of male NOD mice against T1D and that bacterial colonization somehow regulated the production and/or use of testosterone.

To get a sense of the extent to which gut microbiota influenced general physiology of adult male and female NOD mice, the authors used mass spectrometry to profile almost 200 unique small-molecule metabolites in serum. They found that male and female NOD mice raised in standard conditions had distinct profiles of serum metabolites. In contrast, there were few detectable differences between the metabolite profile of males and females raised in germ free conditions. This data suggested two hypotheses: (1) male and female mice had different physiologic responses to the same commensal bacteria, or (2) male and female mice had different commensal communities influencing their hormone and metabolite levels. The authors then sequenced bacterial 16S ribosomal RNA from the intestines of NOD mice at various stages of maturation (just after weaning, at puberty, and as adults). While male and female NOD mice had indistinguishable gut microbiota after weaning, sex-based differences in commensal bacteria were apparent at puberty and became even more pronounced in adulthood.

Having established that adult male and female NOD mice have distinct bacterial populations in their intestines, Markle et al. showed that transplanting the “male” gut microbiota into pre-pubescent female NOD mice altered the composition of the recipient’s commensal populations for several weeks and resulted in increased serum testosterone levels. Importantly, transplantation of male commensal bacteria protected the female recipients from T1D. Markers of T1D disease activity, such as inflammation of beta-islets and production of auto-antibodies, were reduced in recipients of male gut bacteria compared to unmanipulated females. This effect was abrogated upon treatment with the anti-androgen Flutamide, indicating that testosterone levels were a critical regulator of autoimmune pathogenesis.

Markle et al. have put forth an interesting model in which sexual maturation results in sex-specific programming of intestinal commensal bacteria. These distinct populations have differential effects on host physiology and hormonal balance which, in turn, modulate immune function. The centrality of gut microbiota to immunity raises the intriguing possibility of treating a dysfunctional immune system by altering the make-up of the intestine’s commensal communities. Such studies are already underway for intestinal disorders using “fecal transplants”, but the concept may extend to more systemic autoimmune disorders4. This approach would benefit from knowing the specific effects of different bacterial species on host physiology in order to identify the critical components of effective therapeutic microbial regimens.  Similarly, it will be interesting to see which of the changes in metabolite and hormone levels that accompany shifts in commensal populations are most impactful on immune function. The metabolite profiling approach shown in this article could be useful for identifying endogenous compounds that serve as immune modulators.


1. Sex differences in spontaneous versus induced animal models of autoimmunity. Lee TP, Chiang BL. Autoimmun Rev. 2012 May;11(6-7):A422-9. doi: 10.1016/j.autrev.2011.11.020. Epub 2011 Dec 4.

2. Vaccine against autoimmune disease: can helminths or their products provide a therapy? Zaccone P, Cooke A. Curr Opin Immunol. 2013 Mar 2. pii: S0952-7915(13)00027-7. doi: 10.1016/j.coi.2013.02.006.

3. Sex differences in the gut microbiome drive hormone-dependent regulation of autoimmunity. Markle JG, Frank DN, Mortin-Toth S, Robertson CE, Feazel LM, Rolle-Kampczyk U, von Bergen M, McCoy KD, Macpherson AJ, Danska JS. Science. 2013 Mar 1;339(6123):1084-8. doi: 10.1126/science.1233521. Epub 2013 Jan 17.

4. Stool transplants: ready for prime time? Weissman JS, Coyle W. Curr Gastroenterol Rep. 2012 Aug;14(4):313-6. doi: 10.1007/s11894-012-0263-7.


From bedside to bench: linking autoimmunity-associated gene variants to immune function

Autoimmunity results when the immune system, normally tasked to defend against infections and cancer, attacks the body’s own tissues. There are over 80 clinically-distinct autoimmune diseases that differ in terms of which tissues are targeted and which therapies are most effective. Rheumatoid arthritis (RA) and inflammatory bowel disease (IBD) result in the destruction of joints and the intestinal tract, respectively. These diseases respond well to agents such as adalimumab (Humira) and etanercept (Enbrel) that block the action of TNF-alpha, a cytokine that can promote inflammation. During multiple sclerosis (MS) the immune system attacks the central nervous system resulting in progressive neurologic deficits. Despite being an inflammatory disease, multiple sclerosis is actually worsened through the use of anti-TNF-alpha therapies.

Identifying the fundamental dysfunction at the root of an autoimmune disease would aid in choosing the best of available therapies or devising new ones. Advances in genome sequencing technology have allowed researchers to generate an expanding list of genetic differences present in individuals with various autoimmune diseases compared to healthy people. Although several of these disease-associated gene variants have known roles in the immune system, how they contribute to specific autoimmune processes is largely unknown. There is a need for functional characterization of these gene variants in order to determine how they alter immunity and to stratify them as therapeutic targets.

Dr. David Rawlings’ group at Seattle Children’s Hospital sought to address this challenge in a recent paper published in the May 2013 issue of the Journal of Clinical Investigation. The group, with lead author Dr. Xuezhi Dai, investigated a genetic variant of protein tyrosine phosphatase non-receptor 22 (PTPN22) which had previously been linked to several autoimmune diseases, including type 1 diabetes (T1D), RA, Graves’ Disease, and systemic lupus erythematosus (SLE). PTPN22 encodes an enzyme called LYP, a protein tyrosine phosphatase whose general function is to modulate the intensity of certain signals within cellular signaling networks. The disease-linked variant results in an amino acid switch from arginine to tryptophan at position 620 (LYP-R620W). How LYP or LYP-R620W work to modulate immune activity is incompletely understood.

transgenic mouse

To gain insight into the role of LYP-R620W in autoimmune patients, Dai et al. created a genetically engineered mouse with an analogous arginine to tryptophan switch in the mouse version of LYP (called PEP-R619W). The “knock-in” mice expressing PEP-R619W were viable but had slightly shorter life spans compared to their counterparts with normal PEP. As the engineered mice aged they manifested signs of autoimmunity, such as inflamed lung tissue and blood vessels, as well as signs of chronic kidney damage. In addition, PEP-R619W rendered the mice more susceptible to an experimental form of type 1 diabetes.  These mice also produced numerous auto-antibodies, a hallmark of certain autoimmune diseases.

The PEP-R619W knock-in mouse allowed the authors to look in close detail at the effect of this gene variant on specific immune cell populations. Dai et al. found that the knock-in mice had larger numbers of activated/memory T cells than their normal counterparts, indicating a chronically active immune system. T cells from the knock-in mice were shown to be hyper-responsive to stimulation of their antigen receptors indicating augmentation of the intracellular signals that dictate T cell activation. Similarly, the authors found that knock-in mice had larger numbers of specific B cell populations that occur in active immune states. B cells from the knock-in mice proliferated more than those from normal animals in response to stimulation and were more easily induced to secrete antibody. These findings led the authors to conclude that expression of PEP-R619W results in a lower threshold for activation in both T and B cells which contributed to the autoimmune phenotype. Interestingly, the authors discovered that expression of the disease-linked variant exclusively in B cells was sufficient to generate mice with signs of autoimmunity.

Dai et al. provide a great example of how the tools of bench science can be used to deepen the knowledge gained from analysis of patient specimens. Further determination of PEP/LYP substrate specificity and the dynamics of its phosphatase activity during lymphocyte activation could generate targets for the development of highly selective immune suppressants. In addition, the autoimmune phenotype generated with this knock-in mouse is relatively mild. It would be interesting to see how other disease-linked gene variants would cooperate with PEP-R619W to generate either a more aggressive disease or one that resembles a particular autoimmune syndrome. Finally, the ability of B-cell-specific PEP-R619W expression to stimulate autoimmunity suggests that B cells are a critical component of the autoimmune process in patients with this genetic variant. This model provides the opportunity to compare different therapeutic modalities in the PEP-R619W background (for example, B cell depletion versus anti-TNF agents). Such studies could provide the basis for predicting clinical responses to autoimmune therapies based on genotype.


A disease-associated PTPN22 variant promotes systemic autoimmunity in murine models. Dai X, James RG, Habib T, Singh S, Jackson S, Khim S, Moon RT, Liggitt D, Wolf-Yadlin A, Buckner JH, Rawlings DJ. J Clin Invest. 2013 May 1;123(5):2024-36. doi: 10.1172/JCI66963. Epub 2013 Apr 24.

Highlight: How TNF knocks out Tregs!

A healthy and functional immune system requires a delicate balance of pro- and contra-inflammatory signals. Whereas, it is important to induce a strong and efficient immune response against pathogens, it is similarly important to dampen these responses after the pathogen is fought off to revert the immune system to a calm steady state. If the balance is disturbed, diseases can on the one hand, become chronic/overwhelming or, on the other hand, inflammatory responses that cannot terminate can result in autoimmune responses.

Crucial elements in the regulation of excessive immune responses are regulatory T (Treg) cells. Tregs are known to inhibit the response of other immune cells. Their essential role in limiting overwhelming immune responses is demonstrated by the detrimental consequences of their loss. Mice or humans lacking Tregs develop widespread and lethal autoimmune diseases. Besides several surface markers, Tregs are best characterized by the expression of the transcription factor FoxP3. This factor is essential for Treg function and its artificial expression in other T cells can induce a regulatory potential. Therefore, the expression of FoxP3 is required for a T cell to have regulatory potential (Buckner; Josefowicz et al.). However, it was known for many years that in cases of numerous autoimmune diseases FoxP3+ Tregs could be found in high numbers at the sides of inflammation, but that they did not demonstrate any or not sufficient regulatory activity. This enigmatic observation was so far poorly understood (Buckner; Josefowicz et al.).Treg balance

In the March 2013 issue of Nature Medicine Nie and colleagues shed new light on the underlying mechanism that impairs Treg function at the sites of inflammation. Studying Treg cells from rheumatoid arthritis (RA) patients the authors demonstrated that phosphorylation of FoxP3 of the serine at position 418 (S418) is required for its regulatory action. If FoxP3 lacks this particular phosphorylation the Treg cell is not suppressive! FoxP3 S418 in Tregs is usually phosphorylated and hence Tregs are regulatory by default. However, the authors show that due to the action of the enzyme ‘protein phosphatase 1’ (PP1) FoxP3 can lose its S418 phosphorylation. Intriguingly, the presence of the cytokine TNF lead to an up-regulation of PP1 expression in the Tregs in a dose-dependent manner, and this lead to de-phosphorylation of FoxP3 S418. Treg cells expressing a mutant FoxP3 that replaced the serine at position 418 with an alanine retained their suppressive potential even in the presence of TNF, demonstrating the importance of the phosphorylation of S418. With this finding, the authors were able to link the pro-inflammatory milieu (TNF) to a specific effect inside of the Tregs (de-phosphorylation of S418) that lead to the observed loss of the regulatory function of Treg cells. Importantly, the authors were also able to demonstrate the therapeutic potential of this knowledge. They monitored RA patients that underwent treatment with blocking anti-TNF antibodies (infliximab) and found that Tregs from patient PBMCs restored S418 phosphorylation and regained regulatory potential!

This is the second case for a post-transcriptional regulation of FoxP3 that can influence Treg function. Deacetylation of FoxP3 has been linked to impaired Treg function previously (Tao et al.). Additionally, the work of Nie et al. now adds mechanistic information to previous reports on the negative effect of TNF on Tregs (Valencia et al.; Zanin-Zhorov et al.).

Given the ubiquitous role of TNF during inflammation, it is very likely that the mechanism described by Nie et al. applies to many if not all cases of ongoing inflammation where Treg function is impaired. Furthermore, their data on the effects of anti-TNF antibody treatment in RA suggest a similar therapeutic potential in other autoimmune diseases. Surely, this report will ignite further investigation in this direction and will aid the development of better treatments for patients suffering from autoimmune diseases.


Bromberg, J., 2013. TNF-α trips up Treg cells in rheumatoid arthritis. Nat Med, 19(3), pp.269–270.

Buckner, J.H., 2010. Mechanisms of impaired regulation by CD4(+)CD25(+)FOXP3(+) regulatory T cells in human autoimmune diseases. Nat Rev Immunol, 10(12), pp.849–859.

Josefowicz, S.Z., Lu, L.-F. & Rudensky, A.Y., 2012. Regulatory T cells: mechanisms of differentiation and function. Annual Review of Immunology, 30, pp.531–564.

Nie, H. et al., 2013. Phosphorylation of FOXP3 controls regulatory T cell function and is inhibited by TNF-α in rheumatoid arthritis. Nat Med, 19(3), pp.322–328.

Tao, R. et al., 2007. Deacetylase inhibition promotes the generation and function of regulatory T cells. Nature Medicine, 13(11), pp.1299–1307.

Valencia, X. et al., 2006. TNF downmodulates the function of human CD4+CD25hi T-regulatory cells. Blood, 108(1), pp.253–261.

Zanin-Zhorov, A. et al., 2010. Protein kinase C-theta mediates negative feedback on regulatory T cell function. Science, 328(5976), pp.372–376.



Hepatocellular carcinoma (HCC) is an aggressive form of primary liver cancer that occurs more frequently in men than women. This malignancy is different from metastatic liver cancer which originates in another organ (such as the breast or colon) and then spreads to the liver. Even though the incidence of this malignancy is exceptionally high in Asia and Africa, the number of new cases in America and Europe is rapidly increasing, making HCC a worldwide health problem. In spite of improvements in treatment, patients with HCC continue to have a poor prognosis, with 5-year survival rates of only 18%. Therefore, in order to formulate sustained therapeutic strategies, detailed understanding of the molecular network of aggressive HCC is required.

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In addition to significant genomic and proteomic alterations, cancer cells also exhibit highly unique metabolic phenotype which is characterized by increased glucose uptake, enhanced glycolytic activity, decreased mitochondrial activity, low bioenergetic status, and aberrant phospholipid metabolism. This suggests that metabolism may also play a significant role in differentiating normal cells from neoplastic tissues. Several metabolic markers of malignancy are described in particular tumors, such as N-acetyl aspartate and myo-inositol in brain cancers, citrate in prostate cancer, or triglycerides in liposarcomas, based on tissue-specific biochemistry. Cancer metabolite profiling, or cancer metabolomics, is a promising novel approach to help understand the biological events associated with cancer development and progression. A systemic analysis of the pathways in which these genes and biochemical molecules interact may assist in the identification of key biomarkers or drug targets for clinical intervention. Metabolite detection and quantification is usually carried out by nuclear magnetic resonance (NMR) spectroscopy, while mass spectrometry (MS) provides another highly sensitive metabolomics technology.

Using a combination of gene expression and metabolic profile analysis, a recent study by Budhu et al. (2013) reported identification of lipid biomarkers, monounsaturated lipid metabolite (MUPA) and stearoyl-CoA-desaturase (SCD), as key role players in a subset of HCC termed as hepatic stem cell HCC (HpSC-HCC). HpSC-HCC was found to exhibit stem cell–like gene expression traits and associated with poor prognosis as reported by Yamashita and colleagues. By performing metabolomics profiling of tumor and non-tumor tissue samples from 356 patients, Budhu et al. identified 28 metabolites and 169 genes associated with aggressive HCC. Using an integrative data analysis approach to determine gene-metabolite interconnections, this study suggested genes associated with fatty-acid metabolites may play roles in overall survival, stem cell-like HCC and metastasis-related prognosis. Higher expression of one of the genes stearoyl-CoA-desaturase (SCD) was found to be associated with worse survival and disease-free survival. SCD codes for an enzyme responsible for conversion of saturated palmitic acid (SPA) to its monounsaturated form, palmitoleic acid (MUPA). Based on these results, Budhu and colleagues sought to determine the mechanism by which SCD and its related fatty acids, MUPA and SPA, functionally contribute to aggressive HCC and how altering SCD activity may improve this effect. They noted elevated levels of MUPA in aggressive HCCs, and that MUPA enhanced migration and invasion of cultured HCC cells and colony formation by HCC cells, Huh7. Furthermore, HCC cells that had reduced SCD had decreased migration and colony formation in culture and reduced tumorigenicity in mice. Collectively this study suggested that SCD and its related metabolites may be valuable biomarkers and prognostic indicators for molecular re-staging of HCC.



1. Griffin JL, Shockcor JP. Metabolic profiles of cancer cells. Nat Rev Cancer 2004;4:551-61.

2. Griffin JL, Kauppinen RA. A metabolomics perspective of human brain tumours. FEBS J. 2007;274:1132-9.

3. Costello LC, Franklin RB. ‘Why do tumour cells glycolyse?’: from glycolysis through citrate to lipogenesis. Mol Cell Biochem. 2005;280:1-8.

4. Serkova NJ, Glunde K. Metabolomics of cancer. Methods Mol Biol. 2009;520:273-95.

5. Budhu A, Roessler S, Zhao X, et al. Integrated metabolite and gene expression profiles identify lipid biomarkers associated with progression of hepatocellular carcinoma and patient outcomes. Gastroenterology. 2013;144:1066-1075.e1.

6. Yamashita T, Ji J, Budhu A, et al. EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features. Gastroenterology. 2009;136:1012-24.

Considerations for measuring cytokine levels in serum or plasma

Changes in circulating cytokine and chemokine levels have been associated with many human diseases, and thus understanding the relationships between these changes and disease is an important area of medical research.  Circulating levels of these proteins or other chemistries are measured from plasma or serum collected from peripheral blood draws.  It is important to note that the methods of sampling and storage of plasma or serum are critical for accurate measurements.  Here are some important considerations when planning to measure the levels of cytokines and chemokines in serum or plasma.

Blood collection tubes are available in a choice of factors and should blood tubesbe selected based on the analysis being done, as different anti-coagulants support different chemistries.  Plasma is collected from blood drawn into tubes containing anticoagulants, including sodium or lithium heparin which act to inhibit thrombin from blood clotting, or sodium citrate or EDTA which chelate calcium ions to prevent coagulation.  Serum collection tubes contain clot activators, however this method does not allow collection of peripheral blood mononuclear cells (PBMCs) from the same vial, which means that oftentimes, plasma will be the product of choice to maximize the value of blood drawn in a minimal number of tubes from study participants and healthy donors.

luminex service 2Following collection, plasma or serum should be cryopreserved at -80º C.  Cytokines and chemokine levels can be measured by Enzyme-linked immunosorbent assay (ELISA).  However, this method is time consuming and allows measurement of only one factor at a time.  Luminex, a bead-based multiplex assay, can measure up to 100 cytokines, chemokines, or other soluble proteins at a time. Thus, for a given disease cohort, multitudes of measurements can be made from a single small sample of serum or plasma.  Notably, many cytokines and chemokines exist in very low levels in peripheral blood, thus for each cytokine or chemokine to be measured it is important to determine if the detection range of the assay used is sufficient for the known range of circulating levels of that protein.  Also, levels of these proteins may differ depending on whether they were measured in serum or plasma collected in various anticoagulants, so determinations should be done using the most similar methodologies as comparisons.

A methodology paper by de Jager et. al, discusses several important considerations for analyzing cytokine levels from serum or plasma by Luminex.  In this paper, due to unmeasurably low levels of many cytokines, to allow for more dynamic determinations, whole blood was spiked with recombinant cytokines, or treated with LPS for a time period to upregulate expression of cytokines, prior to plasma collection, cryopreservation, and Luminex assays.

One comparison made was the difference in profiles of 15 cytokines in serum, versus plasma from the same donors collected in sodium heparin, EDTA, or citrate.  Overall, cytokine levels were similar with a few exceptions, including IL-6 having the lowest values in serum compared with plasma, while CXCL8 was significantly higher in serum.  The authors concluded that plasma collected in sodium heparin allowed the best measurements overall for the cytokines assessed.  The time it takes to process and store samples after blood collection may also influence cytokine levels and should be done as consistently as possible for the most robust comparisons.

Another hugely important factor is sample storage time.  As with all assays, experimental variation should be minimized, and thus it is common to store plasma or serum samples until the entire cohort has been collected and then analyze all of the samples simultaneously.  This also comes into play when changes in cytokine profiles over time are to be measured from serial samples from an individual. The authors measured cytokine levels from sodium heparin plasma stored at -80º C over time, for up to four years.  Several cytokines including IL-13, IL-15, IL-17 and CXCL8 began to be degraded within one year of storage, while levels of IL-1α, IL-1β, IL-5, IL-6, and IL-10 were degraded by over 50% in 2-3 yearsIL-2, IL-4, IL-12 and IL-18 were much more stable, maintaining their initial levels out to 3 years post initial storage.  Thus, depending on the cytokines being analyzed it is critical to keep these issues in mind.  These are the same issues that are faced with storage of recombinant proteins that are used to generate the ELISA or Luminex standard curves or in other cytokine assays.

Stability of cytokines following several rounds of freeze-thawing were also assessed.  Almost all of the cytokines analyzed with the exception of IL-6 and IL-10 were affected by freeze thawing the samples.  Thus, when storing plasma or serum samples, it is important to freeze the samples in multiple aliquots such that additional assays can be performed while avoiding this issue.

In conclusion, handling and storage of serum and plasma samples as well as the choice of serum versus plasma collected in different anti-coagulants are all important factors to consider when planning for studies that will include measurement of circulating cytokines and chemokines.

Further Reading:

Prerequisites for cytokine measurements in clinical trials with multiplex immunoassays.  de Jager W, Bourcier K, Rijkers GT, Prakken BJ, Seyfert-Margolis V. BMC Immunol. 2009 Sep 28;10:52. doi: 10.1186/1471-2172-10-52.


Deep Brain Stimulation Shows Increased Cerebral Metabolism in Alzheimer’s Patients

Alzheimer’s disease (AD) is the most common form of dementia typically presenting itself after the age of 60, due to protein misfolding in the brain.  According to the National Institute on Aging, currently it is estimated that 5.1 million Americans suffer from AD. Dimentia, is defined as the loss of cognitive function including thinking, remembering, reasoning abilities, and behavioral abilities.  The severity of the disease ranges from pre-dementia (early onset) characterized by small affects on a persons functioning all the way to advanced where a person requires complete dependence on others for daily living.

AD was originally identified by Dr. Alois Alzheimer in 1906 after a brain biopsy of a woman showed abnormal clumps in her brain, now known as Amyloid plaques.  Amyloid plaques originate from amyloid-β (Aβ) deposits which are insoluble fibrous protein aggregates arising from misfolded proteins and polypeptides.

The dominant hypothesis for AD neuropathology is that Aβ plaque formations initiate a cascade that is followed by loss of neurons and synapses in the cerebral cortex and central subcortical regions as well as abnormal levels of neurotransmitters acetylcholine (decreased levels, leading to decreased cognition) and glutamate (increased levels, leading to neuronal over-activation and cell death).  The combined effects being progressive cell death in select regions of the brain. Currently available treatments for AD include drugs that target acetylcholine, glutamate and the amyloid cascade pathway.  These treatments are only efficacious at treating some of the symptoms they don’t stop the underlying decline and death of neurons. Additionally, it is now believed that Aβ plaques are necessary but not sufficient for the cognitive and neurodegenerative effects seen in AD patients. Therefore, the lack of efficacy in currently available treatments and the much needed understanding of the secondary effects due Aβ plaques, shows there is a dire need for safer and more efficacious therapies which can delay and reverse the effects of AD as well as modulate neuronal function in the affected neural circuits.

In a recent TEDx Talk, Dr. Andres M. Lozano from the Toronto Western Research Institute (TWRI) talks about a novel technology used for treating neuronal disorders, deep brain tissue stimulation (DBS). Dr. Lozano’s team have studied the effects of DBS on a variety of neurodegenerative disorders including AD and Parkinson’s disease showing exciting breakthroughs. DBS is surgical procedure where a brain pacemaker is implanted in an affected region of the brain.  The pacemaker can then be controlled externally and directed to send electrical impulses to a specific location.

The human brain consumes about 25% of the total body glucose levels.  In AD patients, it is well known that glucose uptake/metabolism is significantly impaired. Using positron emission tomography (PET) scans with the radiotracer [18F]-2-deoxy-2-fluoro-D-glucose (measures regional cerebral glucose metabolism) Dr. Lozano’s team found that DBS resulted in increased glucose metabolism in AD patients after 1 yr of treatment (http://clinicaltrials.gov/show/NCT00658125). More importantly, the increased metabolism correlated with better outcomes in global cognition, memory, and quality of life.  Based on preclinical studies, it is believed that DBS functions by inducing the generation of new neurons through electrical impulses.  Although, the effects of DBS on Aβ breakdown remain unclear and are under investigation in preclinical models.

It is important to note that not all patients displayed the same level of benefit from DBS.  In addition, all patients in this study had relatively mild AD and higher basal levels of cognitive function and glucose compared to more advanced patients. Therefore, it is possible that the effects of BDS in advanced AD patients will not be as beneficial, although this remains to be determined. It will be interesting to see what the long term effects of DBS will be and whether BDS will be sufficient to keep AD progression at bay.

Further Reading:

Increased Cerebral Metabolism After 1 Year of Deep Brain Stimulation in Alzheimer Disease. Gwenn S. Smith, Adrian W. Laxton, David F. Tang-Wai, Mary Pat McAndrews, Andreea Oliviana Diaconescu, Clifford I. Workman, Andres M. Lozano. Arch Neurol. 2012;69(9):1141-1148. doi:10.1001/archneurol.2012.590.

Memory rescue and enhanced neurogenesis following electrical stimulation of the anterior thalamus in rats treated with corticosterone. Clement Hamani, Scellig S. Stone, Ariel Garten, Andres M. Lozano, Gordon Winocur. Experimental Neurology.Nov;232(1):100-104. doi: 10.1016/j.expneurol.2011.08.023.

Identification of a new HSC viral transduction enhancer, Vectofusin-1

HSC gene therapy is an emerging therapeutic option for several disorders of the blood and immune system.  Ex vivo cell therapies are based on the ability to isolate CD34+ cells from a patient or a normal donor, expansion ex vivo with genetic modification, and systemic administration into the patient following myeloablative treatment.  An efficient method for gene transfer into HSCs is required for successful gene therapy.  Lentiviral vectors (LVs) have emerged as a robust and versatile tool for ex vivo and in vivo gene delivery into multiple cell types, including HSCs.  LVs can either be pseudotyped with viral envelope glycoproteins that confer a broad tropism, such as the vesicular stomatitis virus G (VSV-G) protein, or those that confer a specific HSC tropism, including gibbon ape leukemia virus (GALVTR), feline endogenous retrovirus RD114 (RD114TR), or amphotropic murine leukemia virus (MLV-A) proteins.  However, viral envelopes vary in transduction efficiency.  Thus, transduction protocols often involve the addition of factors to enhance viral entry, including cationic polymers (polybrene) 1 or fibronectin fragments (Retronectin) 2.

gene therapy

Recently, in Molecular Therapy-Nucleic Acids, Fenard et al identified another viral entry enhancer, Vectofusin-1 3.  Vectofusin-1 is a synthetic, histidine-rich cationic amphipathic peptide derived from the LAH4 peptide family.  LAH4 peptides and their derivatives are known to be efficient DNA transfection agents 4.  In this study, the authors examined whether Vectofusin-1 would also enhance gene transfer of LVs into CD34+ cells derived from human umbilical cord blood.  Indeed, Vectofusin-1 significantly increased the transduction efficiency of LVs pseudotyped with various envelopes (VSV-G, GALVTR, RD114TR, MLV), with transduction levels ranging from 50-80% compared to undetectable transduction levels in its absence.  In addition, the increased transduction efficiency was not cytotoxic.  Addition of Vectofusin-1 during transduction of CD34+ cells did not negatively affect subsequent myeloerythroid differentiation in colony-forming cell (CFC) assays in vitro, or hematopoietic reconstitution in immunodeficient BALB-Rag/γC mice in vivo.  The mechanism for the increased transduction efficiency was attributed to insertion of the peptide in the viral and cellular membranes, resulting in an enhancement in both adhesion and fusion of the viral particles with the cell’s plasma membrane.

In short, the authors demonstrated that Vectofusin-1 is a promising LV entry enhancer that can be potentially used in ex vivo transduction of HSCs for subsequent use in clinical applications.  Addition of Vectofusin-1 to the transduction medium had similar effects as the commonly used Retronectin, although the latter is used to coat plates, suggesting a different mechanism of action.  Future experiments will determine whether Vectofusin-1 and Retronectin can be used together to synergistically enhance HSC transduction.


1          Davis, H. E., Morgan, J. R. & Yarmush, M. L. Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption on target cell membranes. Biophys Chem 97, 159-172 (2002).

2          Pollok, K. E. & Williams, D. A. Facilitation of retrovirus-mediated gene transfer into hematopoietic stem and progenitor cells and peripheral blood T-lymphocytes utilizing recombinant fibronectin fragments. Curr Opin Mol Ther 1, 595-604 (1999).

3          Fenard, D. et al. Vectofusin-1, a new viral entry enhancer, strongly promotes lentiviral transduction of human hematopoietic stem cells. Mol Ther Nucleic Acids 2, e90, doi:10.1038/mtna.2013.17 (2013).

4          Kichler, A., Leborgne, C., Marz, J., Danos, O. & Bechinger, B. Histidine-rich amphipathic peptide antibiotics promote efficient delivery of DNA into mammalian cells. Proc Natl Acad Sci U S A 100, 1564-1568, doi:10.1073/pnas.0337677100 (2003).

RORγt+ TREGS: A unique subset of TREGS that specifically promote Colorectal Cancer

tregThe role of CD4+ FoxP3+ regulatory T cells (TREGS) in colorectal cancer (CRC) has continued to be unclear.  TREGS act to suppress inflammatory mechanisms that are associated with tumor progression and can thus act to suppress the development of cancer.  However, TREGS also function to inhibit anti-tumor T cell responses, thereby promoting cancer escape from immune surveillance.  Many studies have been published on the frequencies of TREGS in the peripheral blood and tumors of CRC patients, but there is yet to be a consensus regarding the relationship between TREGS and disease outcome.  In a report by Blatner et. al, the expression of RORγt in a subset of CD4+ FoxP3+ T cells was found to specifically mediate pathogenic pro-tumor activity compared with RORγtCD4+FoxP3+ TREGS in CRC patients.

CD4+ FoxP3+ cells have been classified into three functional populations based on the expression of CD45RA and FoxP3: CD45RA+FoxP3int, CD45RAFoxP3int, and CD45RAFoxP3high.  The CD45RAFoxP3high population exhibits the most suppressive activity of these subsets.  In the study by Blatner et. al, the CD45RAFoxP3high population was found to be specifically expanded in peripheral blood mononuclear cells (PBMCs) and within the tumor of CRC patients and increased with cancer stage.  Because IL-17 expressing CD4+ FoxP3+ cells have been described in the gut and enhanced in patients with CRC and Crohn’s disease, the authors examined CD4+ FoxP3+ populations for expression of the TH17 transcription factor, RORγt. 

In CRC patients, a large fraction of all three subsets of TREGS in PBMCs and in the tumor were found to express RORγt.  Interestingly, when TREG populations were sorted from healthy donors versus CRC patients, CRC patient TREGS  retained suppressive activity over T cell proliferation but had lost their ability to suppress mast cell degranulation.  Expression of IL-17 was also found in a large percentage of CRC TREGS, in a fashion mutually exclusive from IL-10 expression.

To further explore the role of RORγt in CRC, APC∆468 polyposis-prone mice were crossed with mice deficient in RORγt.  RORγt-/-APC∆468 mice were highly resistant to polyp development, had reduced expansion of splenic proinflammatory macrophages, myeloid-derived suppressor cells (MDSCs) and polyp-associated mast cells, compared with RORγt+APC∆468 mice.  Interestingly, the effect of RORγt deficiency in APC∆468 mice was not phenocopied by the loss of IL-17.  Instead, although IL-17 deficiency reduced the frequency of polyps, mast cell recruitment to polyps was enhanced, and ultimately IL-17-/-APC∆468 mice developed invasive lesions.

Overall, this study revealed several fascinating points: CD4+FoxP3+RORγt+ cells appear to be a pathogenic TREG subset that have lost their anti-inflammatory properties and are specifically expanded in CRC patients where they assist in disease progression.  The function of RORγt was not synonymous with IL-17 in TREGS, indicating that other effects of RORγt contribute to the role of these cells in tumor pathogenesis.  Thus, the roles and relationships between FoxP3, RORγt, and IL-17 in TREGS deserve further attention in CRC pathogenesis.  Hopefully, a clearer understanding of this newly identified subset of RORγt+  TREGS and their role in CRC progression will enable much improved methodology for targeting specific TREGS populations in CRC and other disease settings.

Further Reading:

Expression of RORγt marks a pathogenic regulatory T cell subset in human colon cancer.  Blatner NR, Mulcahy MF, Dennis KL, Scholtens D, Bentrem DJ, Phillips JD, Ham S, Sandall BP, Khan MW, Mahvi DM, Halverson AL, Stryker SJ, Boller AM, Singal A, Sneed RK, Sarraj B, Ansari MJ, Oft M, Iwakura Y, Zhou L, Bonertz A, Beckhove P, Gounari F, Khazaie K. Sci Transl Med. 2012 Dec 12;4(164):164ra159. doi: 10.1126/scitranslmed.3004566.

Translational mini-review series on Th17 cells: induction of interleukin-17 production by regulatory T cells.  Afzali B, Mitchell P, Lechler RI, John S, Lombardi G. Clin Exp Immunol. 2010 Feb;159(2):120-30. doi: 10.1111/j.1365-2249.2009.04038.x. Epub 2009 Nov 11.

Inflammation-driven reprogramming of CD4+ Foxp3+ regulatory T cells into pathogenic Th1/Th17 T effectors is abrogated by mTOR inhibition in vivo.  Yurchenko E, Shio MT, Huang TC, Da Silva Martins M, Szyf M, Levings MK, Olivier M, Piccirillo CA. PLoS One. 2012;7(4):e35572. doi: 10.1371/journal.pone.0035572. Epub 2012 Apr 24.

In colorectal cancer mast cells contribute to systemic regulatory T-cell dysfunction.  Blatner NR, Bonertz A, Beckhove P, Cheon EC, Krantz SB, Strouch M, Weitz J, Koch M, Halverson AL, Bentrem DJ, Khazaie K. Proc Natl Acad Sci U S A. 2010 Apr 6;107(14):6430-5. doi: 10.1073/pnas.0913683107. Epub 2010 Mar 22.



Endometrial cancer (EC) is the seventh most commonly diagnosed cancer among women, with 189,000 new cases and 45,000 deaths occurring worldwide each year. In the United States, it is the fourth most commonly diagnosed cancer among women. According to the national Cancer Institute (NCI, USA), in 2013 approximately 50,000 women will be diagnosed with endometrial cancer, with more than an estimated 8,000 deaths from the disease.describe the image

Endometrial cancers are classified into two types: endometrioid (type I) and serous (type II). Type I EC is a less severe form. Risk factors include obesity, anovulation, nulliparity, and exogenous estrogen exposure. This type of EC commonly express both estrogen and progesterone receptors. Clinically, type I EC is more often a low-grade tumor with a favorable prognosis.

On the contrary, type II EC is a more life-threatening form and not associated with estrogen exposure. Clinically, this type of EC is marked by an aggressive clinical course, and has a tendency for early spread and poor prognosis. Endometroid (type I) tumors are treated with adjuvant radiotherapy, whereas serous (type II) tumors are treated with chemotherapy. Even though EC is one of the most common pelvic gynecologic malignancies in the world, to date no targeted therapies are available to treat patients.

Therefore, in order to formulate an efficient treatment plan, detailed genomic characterization of primary and metastatic endometrial cancers are required. Several studies have reported numerous genetic changes associated with endometrial cancer. Type I endometrial carcinomas involve mutations in PTEN, KRAS, FGFR2, PIK3CA and β-catenin, as well as defects in DNA mismatch repair. Type II endometrial carcinomas frequently show aneuploidy and TP53, PIK3CA, and PPP2R1A gene mutations. Using whole exome DNA sequencing on 13 primary serous EC patients, a study by Bell and colleagues (2012) identified high frequency somatic mutations in CHD4, FBXW7, and SPOP genes (associated with chromatin-remodeling and ubiquitin ligase complex). These mutations may play a significant role as driver mutations (gene mutations implicated in cancer initiation and progression) in serous EC.

To better understand the molecular alterations associated with endometrial cancer, a recent study was performed by The Cancer Genome Atlas Research Network (TCGA) using integrated genomic and proteomic analysis appearing in a recent issue of Nature journal (May 2nd, 2013).

Using a multiplatform analysis approach on 373 endometrial carcinomas including low-grade endometroid, high-grade endometroid, and serous carcinomas this study provided key molecular insight into the classification of endometrial cancer. This new study classified endometrial cancer into four new categories:

* The POLE group contained ultrahigh mutation rates in the POLE gene (involved in cellular metabolism) and frequent activation of the WNT/CTNNB1 signaling pathway

* The hypermutated microsatellite instability group showed a high mutation rate, as well as few copy number alterations, and reduced expression of DNA mismatch repair gene MLH1

* The copy-number low group showed increased expression of progesterone receptor and DNA repair protein RAD50

* The copy-number high group composed of mostly serous tumors and serous-like endometroid tumors and exhibited increased transcriptional activity of cell cycle related genes (MYC, CCNE1, PIK3CA, CDKN2A etc.) and a mutation in tumor suppressor gene TP53.

In addition, this study also observed compelling similarities in the molecular phenotype between 25% of high-grade endometroid tumors and uterine serous carcinoma, suggesting that this genome-based molecular characterization may benefit these patients. Overall, this new molecular characterization might facilitate the discovery of effective, targeted treatments as well as may affect post-surgical adjuvant treatment for women with endometrial cancer.


Bansal N, Yendluri V, Wenham RM (2009) The molecular biology of endometrial cancers and the implications for pathogenesis, classification, and targeted therapies. Cancer Control 16: 8-13.

Hecht JL, Mutter GL (2006) Molecular and pathologic aspects of endometrial carcinogenesis. J Clin Oncol 24: 4783-4791.

Kandoth C, Schultz N, Cherniack AD, Akbani R, Liu Y, Shen H, Robertson AG, Pashtan I, Shen R, Benz CC, Yau C, Laird PW, Ding L, Zhang W, Mills GB, Kucherlapati R, Mardis ER, Levine DA, Network CGAR (2013) Integrated genomic characterization of endometrial carcinoma. Nature 497: 67-73.

Kuhn E, Wu RC, Guan B, Wu G, Zhang J, Wang Y, Song L, Yuan X, Wei L, Roden RB, Kuo KT, Nakayama K, Clarke B, Shaw P, Olvera N, Kurman RJ, Levine DA, Wang TL, Shih IM (2012) Identification of molecular pathway aberrations in uterine serous carcinoma by genome-wide analyses. J Natl Cancer Inst 104: 1503-1513.

Le Gallo M, O’Hara AJ, Rudd ML, Urick ME, Hansen NF, O’Neil NJ, Price JC, Zhang S, England BM, Godwin AK, Sgroi DC, Hieter P, Mullikin JC, Merino MJ, Bell DW, Program NISCNCS (2012) Exome sequencing of serous endometrial tumors identifies recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes. Nat Genet 44: 1310-1315.