Development of Anti-viral CD4+ T cells in unexposed individuals

HIVIn a study published in the February 2013 issue of Immunity, Su. et. al., characterized the CD4+ T cell repertoire from adult human peripheral blood mononuclear cells (PBMC) and made a fascinating observation: that memory T cells can develop against pathogens the host has never been exposed to, including HIV-1, cytomegalovirus (CMV), and herpes simplex virus (HSV).

To determine the baseline frequencies of pathogen and self-antigen-specific CD4+ T cell populations in naive individuals by flow cytometry, PBMCs were stained with major histocompatibility complex (MHC)-peptide tetramers for HLA-DR4 -restricted epitopes.  The MHC-peptides assessed included peptides from viral pathogens including HIV-1 (gag p24), CMV (pp65), and HSV (VP16), as well as self-peptides: the melanoma-associated antigen gp100, the arthritis-associated antigen fibrinogen, and the diabetes-associated antigen preproinsulin. For both self and pathogen-associated antigens, the frequency of tetramer-positive cells was found to be one to ten cells per million CD4+ T cells, in individuals previously unexposed to these viruses.  Prior exposure to viral pathogens was determined by highly sensitive serological tests.

Interestingly, on average across individuals, greater than 50% of these antigen-specific CD4+ T cells were found to be memory T cells based on CD45RO+ staining and expression of CD4+ memory T cell-associated genes.  Furthermore, viral antigen-specific CD45RO+ memory T cells secreted IFNg when stimulated with phorbol myristate acetate (PMA) plus ionomycin.  Sequencing of the TCR-beta chain from naïve and memory HIV-1-specific CD4+ T cells indicated that the antigen-specific memory phenotype but not naive CD4+ T cell populations had arisen from clonal expansion.  Thus the memory phenotype of these antigen-specific T cells indicates prior antigen experience and subsequent clonal expansion.

The question remained though, as to how individuals had developed CD4+ memory T cell responses against pathogens they had not apparently been exposed to.  One hypothesis for the existence of these CD4+ memory T cell populations is development via cross-reactivity to other antigens.  BLAST analysis of the HIV-1 peptide sequence identified several similar sequences present in gut and soil-resident bacteria, marine algae, and plants.  HIV-1-specific CD4+ memory T cell clones from various individuals showed cross-reactivity to several of these environmental microbial antigens as measured by production of IFNg and IL-2, and proliferation when stimulated with MHC-peptides.  Interestingly, antigen-specific CD4+ memory T cells elicited by vaccination of individuals with an H1N1 influenza virus vaccine were reciprocally cross reactive to several other microorganisms.

Overall, this study shows that pathogen-specific CD4+ memory T cells commonly arise in individuals through exposure to other environmental microbes containing similar peptide sequences.  The question remains however, as to whether these cross-reactive antigen-specific T cells are able to exhibit protective immune responses.


Further Reading:

Virus-Specific CD4 (+) Memory-Phenotype T Cells Are Abundant in Unexposed Adults.  Su LF, Kidd BA, Han A, Kotzin JJ, Davis MM. Immunity. 2013 Feb 5. pii: S1074-7613(13)00052-6.

Derivation and maintenance of virtual memory CD8 T cells.  A.D. Akue, J.Y. Lee, S.C. Jameson.  J. Immunol., 188 (2012), pp. 2516 – 2523

Nonrandom attrition of the naive CD8+ T-cell pool with aging governed by T-cell receptor:pMHC interactions. B.D. Rudd, V. Venturi, G. Li, P. Samadder, J.M. Ertelt, S.S. Way, M.P. Davenport, J. Nikolich-Žugich.  Proc. Natl. Acad. Sci. USA, 108 (2011), pp. 13694–13699.

Maintenance of peripheral naive T cells is sustained by thymus output in mice but not humans.  I. den Braber, T. Mugwagwa, N. Vrisekoop, L. Westera, R. Mögling, A.B. de Boer, N. Willems, E.H. Schrijver, G. Spierenburg, K. Gaiser et al.  Immunity, 36 (2012), pp. 288–297

Photo credit: Microbe World / / CC BY-NC-SA

New Clues for Chemotherapy-Induced Cognitive Impairment

Cancer is the formation of a malignant neoplasm, initiated by a cell that escaped apoptosis upon which mutations during DNA replication were not repaired; this cell, no longer regulated, continuously proliferates while each progeny will carry the previous mutation(s), while generating new ones, eventually resulting in a population of unregulated cells (malignant tumors) that will metastasize and take over the host’s body.  Thus, the conventional cancer treatment consists of surgical resection of the tumor mass, followed by administration of agents which kill any diving (mitotic) cells in the body (chemotherapy and radiation therapy). Consequently, these agents will not only target proliferating cancer cells, but they also destroy healthy proliferating cells, hence the side effects associated with chemotherapy, such as hair loss, nausea and cognitive deficits. This article emphasizes on chemotherapy’s role in disrupting processing speed, working memory and attention in humans.

Adult neurogenesis is the post-natal process of generating functional neurons (and glial cells) from adult neural precursors/progenitor cells (NPCs) throughout life. The two regions in the adult brain in which neurogenesis occurs are the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus. Newly generated cells in the SGZ can differentiate into functional neurons and integrate into the adult hippocampus’s DG as granule cells. Granule cells are involved in memory formation and many aspects of learning, with the exception of long-term memory storage.

Neurogenesis,chemotherapy,cancer,memory loss

Over 50% of cancer patients undergoing chemotherapy report significant cognitive impairment and declines in their overall cognitive processing, collectively referred to as “chemo-brain”. Thus, chemotherapy-induced loss of newly generated neurons in the hippocampus and impeding adult neurogenesis as the cause of such cognitive decline is a compelling notion. Furthermore, one of the highly “cognitive” oscillations in the human brain is the theta rhythm, which is mainly generated in the hippocampus and is also associated with processes of learning and memory. This rhythmic slow activity is also the most efficient synchronized electroencephalographic (EEG) activity that can be recorded from the brain.  It has been suggested that since synchronized oscillatory activity implements communication between functionally related structures during the process of learning, a chemotherapy-induced disruption in theta activity may obstruct inter-regional communication and result in learning deficits.

In a recent study published in the European Journal of Neuroscience, Shors’s group reported that prolonged systemic chemotherapy disrupts both the structural and functional integrity of the hippocampus, resulting in highly specific learning impairments. Their results show that chemotherapeutic agents instigate the learning deficits described in ‘chemo-brain’ via decreases in hippocampal adult neurogenesis and theta activity. Interestingly however, they are not responsible for the disruption of the hippocampus-independent memory for previously (pre-treatment) learned associations. In this study, the effects of chemotherapy on hippocampal adult neurogenesis, theta activity and learning were investigated through evaluating associative learning in adult male Sprague–Dawley rats by recording the hippocampal local-field potentials after several weeks of cyclic administration of the chemotherapeutic agent temozolomide (TMZ).The results revealed that TMZ’s effects on learning and theta activity were specific to a task in which an association had to be formed between temporally related but separate events, while no affects were observed in the expression of an already acquired trace memory.


TMZ is a small lipophilic monofunctional DNA alkylating agent, commonly used to treat metastatic malignant melanomas as well as tumors of the central nervous system (CNS), such as Glioblastoma Multiforme (GBM). Shors’s group also showed evidence of TMZ’s selective affect on neurogenesis, and not glia generation. They proposed the reason for this observation to be the possible differences in DNA repair mechanisms between neural precursors and glia. This notion is further supported by previous reports indicating that unlike TMZ, chemotherapeutic agents that do not readily cross the blood brain barrier (BBB), lower hippocampal neurogenesis and give rise to abnormal dendritic morphology. Additionally, it has been shown that cells surviving radiation therapy tend to differentiate into glial cells rather than neurons.

While most cancer patient undergoing chemotherapy experience short-term memory loss and difficulty performing complex tasks, about 15% of patients experience long-lasting cognitive problems due to long-term chemotherapy treatment. Identifying the underlying cause of some of these cognitive deficits is a major step towards finding of alternative agents or modifying current ones to eliminate or alleviate these issues.

Further Readings: 

Chemotherapy Disrupts Learning, Neurogenesis and Theta Activity in the Adult Brain.

Cognitive Side Effects of Cancer Therapy Demonstrate a Functional Role for Adult Neurogenesis.

Changing the Rate and Hippocampal Dependence of Trace Eyeblink Conditioning: Slow Learning Enhances Survival of New Neurons.


Thyroid cancer is a form of tumor growth located within the thyroid gland. It is the most prevalent endocrine malignancy and affects both men and women at any age. During the early stages of the disease, many patients do not experience symptoms. However, as the cancer progresses, symptoms can include a lump or nodule in the front of the neck, hoarseness or difficulty speaking, swollen lymph nodes, difficulty swallowing or breathing, and pain in the throat or neck. There are different types of thyroid cancer: papillary, follicular, medullary, anaplastic, and variants. Among these, differentiated thyroid carcinoma, namely papillary and follicular thyroid carcinoma, makes up about 94% of these cases.

Radioactive iodine (RAI) treatment is given to treat differentiated thyroid carcinoma. The treatment uses a radioactive form of iodine called iodine 131 or I-131. The RAI circulates throughout the body in the bloodstream. Thyroid cancer cells pick up the RAI wherever they are present in the body. The radiation in the iodine then kills the cancer cells. This is a targeted treatment. It doesn’t affect other body cells, because only thyroid cells take up the RAI.  Even though differentiated thyroid cancer is curable, recurrence occurs in 20-40% patients. In about 5% patients with cellular dedifferentiation, the disease develops more aggressive behavior and metastatic growth. This results in tumor resistance to conventional therapy, RAI uptake, and poor prognosis. Resistance to the RAI therapy has been reported to be responsible for a large number of deaths in advanced thyroid carcinomas (papillary and follicular), and represents a major clinical challenge.

Approximately 70% papillary thyroid carcinoma is typically associated with mutations in RET, describe the imageRAS and BRAF oncogenes. In follicular thyroid carcinoma mutations of RAS oncogene in addition to other gene mutations was also observed. Aberrant activities of these oncogenes results in constitutive activation of the MAPK-pathway leading to inhibition of sodium-iodide symporter and thyroid peroxidase genes which participate in iodide uptake and thyroid hormone production respectively. In a pre-clinical study, Chakravarty et al. (2011) showed that mice with poorly differentiating thyroid cancer and overexpressing BRAF (V600E) oncogene failed to uptake RAI. Shutting BRAF activation off or inhibiting the MAPK-pathway with kinase inhibitors targeting BRAF or MEK rendered mice susceptible to a therapeutic dose of RAI. This suggests that inhibition of the MAPK-pathway and its associated protein kinases with the protein kinase inhibitors may facilitate RAI uptake in refractory thyroid cancer patients harboring MAPK-pathway activation.

A pilot clinical study published recently in The New England Journal of Medicine (368;7 February 14, 2013) by  Ho et al. observed that inhibition of the MAPK pathway with MEK inhibitor selumetinib (AZD6244) enhanced RAI uptake in thyroid cancer patients that are CT scanresistant to RAI. Selumetinib is currently in clinical trials for various solid and hematologic malignancies. Among 24 patients screened for the study 5 had NRAS-mutant tumors. All of them showed augmented RAI uptake following treatment with selumetinib; 4 patients exhibited confirmed partial responses (PR) and in 1 patient no disease progression was noted following RAI treatment. No significant levels of toxic effects to selumetinib were observed in this study. Increased RAI uptake and confirmed PR was also observed in 1 patient with BRAF mutation after treatment with selumetinib. However, selumetinib treatment in majority of the patients with BRAF mutations enrolled in this study did not increase RAI uptake up to the threshold level required for therapy. Therefore, further studies are required to understand the differences observed between RAS-mutant and BRAF-mutant tumors.

In addition to the activation of oncogenes, overexpressions of many tyrosine kinase receptors (c-Met, EGF, VEGF etc) has also been reported in dedifferentiated thyroid cancers. The signaling cascades of these receptors cause constitutive activation of both MAPK and PI3K/Akt pathway and thereby disrupting iodine transport and thyroid hormonogenesis. This further suggests that pharmacological abrogation of the MAPK pathway and also PI3K/Akt pathway may be clinically beneficial in RAI-refractory thyroid cancer. Several clinical trials are currently evaluating efficacy of various protein kinase inhibitors (GSK2118436, Sorafenib, Everolimus, Lenvatinib) targeting these pathways to re-sensitize RAI-refractory thyroid carcinomas to radioactive iodine therapy.


Suggested reading:

1. Chakravarty, D., Santos, E., Ryder, M., Knauf, J. A., Liao, X. H., West, B. L., Bollag, G., Kolesnick, R., Thin, T. H., Rosen, N., et al. (2011). Small-molecule MAPK inhibitors restore radioiodine incorporation in mouse thyroid cancers with conditional BRAF activation. J Clin Invest 121, 4700-4711.

2. Ho, A. L., Grewal, R. K., Leboeuf, R., Sherman, E. J., Pfister, D. G., Deandreis, D., Pentlow, K. S., Zanzonico, P. B., Haque, S., Gavane, S., et al. (2013). Selumetinib-enhanced radioiodine uptake in advanced thyroid cancer. N Engl J Med 368, 623-632.



Autoimmunity in Active Relapsing-Remitting Multiple Sclerosis

Multiple sclerosis (MS) is a chronically progressive, neuroinflammatory autoimmune disease of the central nervous system (CNS), mediated in part by CD4+ T-cells, which have escaped regulation and recognize myelin protein peptides.

CD4+ CD25+ regulatory T-cells (Tregs) are a subpopulation of suppressor T-cells that play a major role in maintenance of peripheral immune tolerance by active suppression of potential auto-aggressive T-cells. In contrast to patients with secondary progressive MS (SPMS) who have normal Treg function, patients with relapsing-remitting MS (RRMS) have functionally impaired Tregs; this lack of regulatory suppression leads to the infiltration of pathogenic CD4 T-cells  into the CNS and the subsequent neuroinflammation.

Although patients with RRMS have a lower Treg number and function, previous studies have shown no correlation between therapeutic response and increased Treg number. However, based on data obtained from several autoimmune animal models, it has been speculated that the resistance of pathogenic CD4+ effector T cells (Teffs) to suppression by Tregs may be responsible for the failed tolerance in autoimmunity. Moreover, Teff resistance has been reported in some human autoimmune diseases such as type 1 diabetes mellitus (T1D), rheumatoid arthritis (RA), and psoriasis. Teff resistance is stimulated by several factors, including tumor necrosis factor–α (TNF-α), interleukin-4 (IL-4), IL-12, IL-6, IL-7, IL-15, IL-21 and the maturation state of CD4 T-cells.

In a recent study published in Nature, Schneider’s group demonstrated the presence of Teff resistance in individuals with aggressive RRMS and the role of interleukin-6 (IL-6) in promoting Teff resistance to Tregs.

MS,CD4 T-cells,effector,regulatory

Previous studies have shown the implication of IL-6 in MS pathology; IL-6 has been shown to inhibit apoptosis in T-cells, it is required for the differentiation of T-helper 17 (TH17) cells, and local exposure to IL-6 can result in the development of Teffs resistant to suppression. During establishment of an immune response-derived inflammation, IL-6 levels elevate rapidly and bind to the IL-6 receptor α (IL-6Rα) on the CD4 T-cell’s surface. Next Glycoprotein 130 (gp130) is recruited to this IL-6-IL-6R complex, which results in activation and phosphorylation of the signal transducer and activator of transcription 3 (STAT3). Otherwise, IL-6 cytokine can bind soluble IL-6Rα (sIL-6Rα) in the serum and induce the phosphorylation of STAT3 by forming a complex that signals through membrane-bound gp130. In addition to the genetic correlation between variants in the STAT3 locus and MS susceptibility, a significant increase in phosphorylated STAT3 (pSTAT3) as well as IL-6Rα expression on CD4+ T cells, have been reported in RRMS patients.

In Schneider’s recent study, the role of Teff resistance in RRMS’s failed tolerance was investigated by comparing Teffs from RRMS patients and healthy individuals, via Treg suppression assays; the obtained results show that Teff resistance is present only in the Teffs of RRMS patients with active disease (two or more clinical exacerbations or presence of one or more gadolinium-enhancing lesions on MRI within 2 years of sampling) and not those with inactive/mild disease.

Furthermore, by performing suppression assays in the presence of the STAT3 inhibitor (blocking STAT3 phosphorylation), they observed enhanced suppression, indicating a positive correlation between the degree of Teff resistance and increased pSTAT3 in response to IL-6; their data imply that an increase in IL-6Rα expression on CD4+ T-cells and enhanced IL-6mediated phosphorylation of STAT3 are major contributors to the impaired suppression observed among their RRMS subjects. They hypothesized that in active RRMS patients, the increased pSTAT3 and resistance of the pathogenic CD4 T-cells to regulation mediated by Tregs, is due to the elevated IL-6 production by microglia, astrocytes, endothelial cells, neurons, oligodendrocytes, or infiltrating T-cells in the CNS.

T-cells,autoimmune,Multiple Sclerosis,MS

Schneider’s new findings suggest utilization of IL-6Rα expression and IL-6 mediated pSTAT3 as new therapeutic markers for determining disease activity as well as evaluating responsiveness to immunomodulatory therapies, such as tocilizumab (an IL-6Ra antagonist) in RRMS. Another significant aspects of this study is the unconventional technical approaches utilized in assessing the impact of IL-6 on suppression within an antigen-presenting cells (APCs)-free system, as well as ensuring consistency of activation and source of Tregs via a bead-based stimulation assay and in vitro–generated Tregs respectively. These unique techniques are key to the conclusions drawn from this study and useful for future MS research.


Further Reading:

In Active Relapsing-Remitting Multiple Sclerosis, Effector T Cell Resistance to Adaptive Tregs Involves IL-6-Mediated Signaling.   

In vitro Treg Suppression Assays.

How Cardiosphere-derived Cells Regenerate the Injured Heart

According to the Centers for Disease Control, approximately one million heart attacks (Myocardial Infarction, or MI) occur per year in the U.S. [1].  Loss of functioning heart muscle due to an MI can result in congestive heart failure which is the leading cause of death and disability inAmerica.  It is estimated that about half of those with congestive heart failure die within five years of diagnosis [1].  New therapies are desperately needed to treat and prevent the clinical complications that follow a heart attack. Unfortunately, the heart muscles (cardiomyocytes) do not ordinarily regenerate once damage has occurred by MI and thus many researchers are looking into stem cell therapies for heart failure and disease.

The widely accepted view that the heart was not capable of regeneration was established in 1925 by Karsner et al. who demonstrated that the heart grew larger due to cardiac hypertrophy (increase in cell size) as opposed to hyperplasia (increase in cell number) [2].  However, in 2009, Bergmann et al. demonstrated that the human heart was capable of self-renewal by measuring the age of cardiomyocytes in individuals exposed to carbon-14 generated by nuclear bomb tests during the Cold War [3].  The work of Hsieh et al. showed that adult heart regeneration was due to an actual subset of progenitor cells as opposed to increased proliferation of resident cardiomyocytes [4].  This group used an α-myosin heavy chain Cre-Lox transgenic mouse model (Mer-CreMer-ZEG mouse), where 100% of the cardiomyocytes are b-galactosidase (b-gal)  positive but activation of Cre recombinase by tamoxifen results in 80% EGFP (Enhanced Green Fluorescent Protein) positive cardiomyocytes and 20% b-gal positive cardiomyocytes.  After myocardial infarction there was a 15% increase in b-gal positive cardiomyocytes indicating that cardiomyocytes were being generated from stem/ progenitor cells, which were never exposed to Cre recombinase. Thus, while there is insufficient capacity of mammalian adult heart tissue to undergo self-repair, injured myocardium can increase cardiomyocyte generation from non-myocyte precursors.

While protocols aimed to mobilize endogenous stem/ progenitor cells to sites of injury or disease would be greatly beneficial to cardiac patients, transplantation of autologous or allogeneic cells is also a promising alternative.  Cell therapies may result in phenotypic replacement of heart cells, reduce scar formation, increase survival and function of endogenous cardiac cells by increasing in vivo bioavailability of growth factors, and exert immunomodulation by releasing soluble molecules and express immune-relevant receptors (chemokine receptors and CAMs (cellular adhesion markers)).  The first clinical trial using a stem cell therapy for cardiac disease was performed over a decade ago using autologous bone marrow cells (BMCs) in patients after acute MI which reported improved heart function and myocardial perfusion [5].  Since then, numerous adult stem cell therapies targeting cardiac disease have been launched with the majority of therapies involving BMCs [6].

describe the imageRecently, a phase I clinical trial based on administration of autologous cardiosphere-derived cells (CDCs) to assess safety in patients with left ventricular dysfunction after MI was completed by Dr. Eduardo Marban atCedars-SinaiMedicalCenter[7].  The formation of cardiospheres was first described by Messina’s group who showed that human and mouse heart explants generated a layer of fibroblast-like cells over which small, phase-bright cells migrated, and once these phase-bright cells are transferred to non-adherent plates, they generate three-dimensional spherical structures [3].  Marban’s group modified Messina’s protocol by placing the cardiospheres in adherent plates where the cells begin to grow in monolayer, hence cardiosphere-derived cells, allowing for easier and faster expansion.  Promising preclinical data which showed a reduction in infarct size and improved cardiac function after transplantation of CDCs in a porcine animal model prompted the phase I clinical trial [8].

This trial, known as CADUCEUS (CArdiosphere-Derived aUtologous stem CElls to reverse ventricUlar dySfunction), enrolled patients 2-4 weeks after MI (with left ventricular ejection of 25-45%) who received either standard of care (no intervention, usual medical management after MI), low cell dose (12.5 million) or high cell dose (25 million) of autologous CDCs ( NCT00893360).  Percutaneous endomyocardial biopsies were used to obtain the heart tissue necessary for generation of the predetermined dose of autologous CDCs (usually within 36 days of sampling) and cells were delivered through an angioplasty catheter in the infarct-related artery. Results from the trial showed that the CDCs were relatively safe: no patients died, developed cardiac tumors or experienced a major adverse cardiac event (MACE).  Preliminary efficacy suggested that patients treated with CDCs had reductions in scar mass and increases in viable heart mass as measured by magnetic resonance imaging (MRI), as well as increases in regional contractility and systolic wall thickening at 12 months compared to controls [7].  However, the trial did not confirm whether there was true increased myocardial mass, whether the CDCs themselves could possibly distort the myocardial architecture and/or how the CDCs potentially could regenerate the injured heart.

Recently Marban’s group has shed light on the potential mechanism of how the CDC therapy reduces scarring and regenerates healthy tissue after an MI, published in EMBO Molecular Medicine [9].  They applied lineage tracing systems using the Mer-CreMer-ZEG mouse with additional labeling tools to investigate the cellular origins of regeneration in an adult mouse after surgical induction of MI followed by intramyocardial injection of mouse CDCs.  Impressive results revealed that after MI new cardiomyocytes arise from both pre-existing cardiomyocytes and undifferentiated progenitors and that transplanted CDCs further up-regulate host cardiomyocyte proliferation and recruitment of endogenous progenitors to the infarct site.  The increase in cardiomyocyte proliferation and progenitor recruitment was accompanied by structural and functional changes in the infracted heart, specifically decreased scar size, increased infracted wall thickness and myocardium (cardiomyocyte hypertrophy was excluded), and increased cardiac function.  Interestingly, these effects occurred despite robust CDC engraftment indicating that CDCs do not contribute to phenotypic replacement but promote heart regeneration by indirect means.  These promising findings indicate that stem cell therapies may stimulate dormant surviving cells after injury and boost natural repair mechanisms.

However, there still remain many challenges for developing cell therapies for cardiac disease.  First, the technology is still immature.  With autologous approaches, patient-to-patient variability can arise such as purity and potency of the cells.  Harvesting the source material, obtaining a sufficient quantity of cells, delivery of the end product and the optimal site of delivery will need to be established.  In the future, Marban’s group plans on using allogeneic CDCs obtained from donor organs, which allows for an off-the-shelf-therapy, to treat patients following MI.  A thorough cell biological characterization of the CDCs will be required to understand the molecular identity and mechanism(s) of action of these cells.  Second, the underlying mechanism(s) of cardiac repair and/ or regeneration after MI remain elusive.  Additionally, treating chronic or congestive heart failure with the aim of generating new contracting heart muscle is more complex.  Third, it is difficult to make conclusive statements about the results obtained from many of the clinical trials since they have small patient size and therefore limited statistical power.  It is unclear how the findings will generalize to a larger population of patients.  Fourth, most of the clinical trials use surrogate endpoints which are measures of an effect of a certain treatment expected to predict clinical benefit (or harm, or lack of benefit or harm) such as measuring cardiac function.  Even though surrogate endpoints may correlate with a real clinical endpoint, they do not have a guaranteed relationship.  Therefore, clinically meaningful endpoints such as improvement in overall survival and prevention of future heart failure are needed.  In the CADUCEUS trial there was only enhanced regional structure/ function but no significant effect on global functional endpoints such as ejection fraction.  A phase II double-blinded placebo-controlled clinical trial will need to be performed to assess true efficacy.  It will be several more years before we have a clear understanding of the true potential of cell therapy in cardiac disease.  In the meantime, studies to understand the cellular sources and underlying cellular mechanisms involved in cardiac regeneration are still needed.



1.         CDC. Heart Disease Facts.  updated October 16, 2012; Available from:

2.         Karsner, H.T., O. Saphir, and T.W. Todd, The State of the Cardiac Muscle in Hypertrophy and Atrophy. Am J Pathol, 1925. 1(4): p. 351-372 1.

3.         Carvalho, A.B., B.K. Fleischmann, and A.C. Campos de Carvalho, Cardiac Stem Cells, in Resident Stem Cells and Regenerative Therapy, R.C.d.S. Goldenberg, Editor. 2012, Academic Press: Waltham, MA. p. 141-155.

4.         Hsieh, P.C., et al., Evidence from a genetic fate-mapping study that stem cells refresh adult mammalian cardiomyocytes after injury. Nat Med, 2007. 13(8): p. 970-4.

5.         Strauer, B.E., et al., Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation, 2002. 106(15): p. 1913-8.

6.         Sheridan, C., Cardiac stem cell therapies inch toward clinical litmus test. Nat Biotechnol, 2013. 31(1): p. 5-6.

7.         Makkar, R.R., et al., Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial. Lancet, 2012. 379(9819): p. 895-904.

8.         Johnston, P.V., et al., Engraftment, differentiation, and functional benefits of autologous cardiosphere-derived cells in porcine ischemic cardiomyopathy. Circulation, 2009. 120(12): p. 1075-83, 7 p following 1083.

9.         Malliaras, K., et al., Cardiomyocyte proliferation and progenitor cell recruitment underlie therapeutic regeneration after myocardial infarction in the adult mouse heart. EMBO Mol Med, 2013. 5(2): p. 191-209.

Identification of CD8+ TC1, TC2, and TC17 populations in human PBMC

Human peripheral blood mononuclear cells (PBMC) are composed of heterogeneous populations of various immune cell types.  CD4+ and CD8+ T cells are known to exist in various functional and differentiated states.  Following antigen experience, naïve T cells are thought to progressively differentiate along a path through central memory, effector memory, and terminally differentiated effector states. Markers for differentiating PBMC T cells into these subtypes using multiparametric flow cytometry include: CD3, CD4, CD8, CD45RA or CD45RO, and CCR7 or CD62L.

The cytokine milieu T cells are exposed to during antigen encounter directs differentiation into various subtypes that exhibit unique functional properties and gene expression programs including cytokines, transcription factors, and surface markers.  This is true for both CD4+ and CD8+ T cells. In a previous post, I discussed various markers that can be utilized by flow cytometry to identify CD4+ TH1, TH2, and TH17 populations in human PBMC.

CD8+ T cells can also differentiate into unique subsets similar to TH1, TH2, and TH17 CD4+ T cells.  The CD8+ versions of these subsets are referred to as TC1, TC2, and TC17 CD8+ T cells, respectively, and are defined by expression of the same characteristic cytokines as their CD4+ counterparts.

As previously discussed, expression of subset-specific surface markers is easily determined by flow cytometry. Identification of intracellular cytokine production in T cells can be assessed following 4-6 hours of TCR stimulation with anti-CD3 and anti-CD28 antibodies or the combination of Phorbol 12-Myristate 13-Acetate (PMA) and ionomycin in the presence of brefeldin A or monensin.  The cells are then fixed and permeabilized with buffers such as BD Biosciences’ Cytofix Cytoperm buffer set followed by antibody staining for cytokine expression and flow cytometry.

Like CD4+ TH1 cells, CD8+ TC1 cells characteristically produce IFNγ.  This population is by far the most common cytokine-producing CD8+ cell subset, and is very easy to identify using intracellular staining for IFNγ.

As with CD4+ TH2 cells, CD8+ TC2 cells can be identified by expression of IL-4, IL-5, and IL-13Cosmi et. al, found that the surface marker CRTH2 was a robust marker for identification of CD8+ and CD4+ cells producing IL-4, IL-5, and IL-13 expression but not IFNγ.  Expression of chemokine receptors CCR3 and CCR4 however, did not exclude IFNγ-producing cells.  Because expression of IL-4, IL-5, and IL-13 can be difficult to detect, CRTH2 may be the easiest of these markers for TC2 identification in human PBMC.

CD8+ TC17 cells are characterized by expression of the cytokine IL-17.  Expression of the chemokine receptors CCR5 and CCR6 were shown to enrich for IL-17 producing CD8+ cells.  However CCR5 and CCR6 expression are also associated with TC1 cells, and thus may not be useful to differentiate between these subsets.

CD8 Tc1 Tc2 Tc17 PMA resized 600

Figure: Expression of IFNγ, IL-17, and CRTH2 in CD8+ T cells from PBMC stimulated with for 4 hours with PMA/ionomycin.

In my own studies, I have utilized TCR or PMA/ionomycin stimulation of PBMCs to successfully identify IFNγ (TC1) and IL-17 (TC17) expressing cells, and CRTH2 expression to identify TC2 cells, as these may be the most robust markers for identification of these unique CD8+ T cell populations. Note also that these same markers reliably detect CD4+ TH1, TH17, and TH2 cells, respectively. Thus, these markers are useful to quantitate and study the function of both CD8+ and CD4+ subsets in human PBMC.


Additional Reading

Generation of polarized antigen-specific CD8 effector populations: reciprocal action of interleukin (IL)-4 and IL-12 in promoting type 2 versus type 1 cytokine profiles.  Croft M, Carter L, Swain SL, Dutton RW. J Exp Med. 1994 Nov 1;180(5):1715-28.

CRTH2 is the most reliable marker for the detection of circulating human type 2 Th and type 2 T cytotoxic cells in health and disease.  Cosmi L, Annunziato F, Galli MIG, Maggi RME, Nagata K, Romagnani S.  Eur J Immunol. 2000 Oct;30(10):2972-9.

Cutting edge: Phenotypic characterization and differentiation of human CD8+ T cells producing IL-17.  Kondo T, Takata H, Matsuki F, Takiguchi M. J Immunol. 2009 Feb 15;182(4):1794-8.

Functional expression of chemokine receptor CCR6 on human effector memory CD8+ T cells.  Kondo T, Takata H, Takiguchi M. Eur J Immunol. 2007 Jan;37(1):54-65.

Overcoming Resistance to Tyrosine Kinase Inhibitors in CML

Chronic myeloid leukemia (CML), a form of slowly progressing blood and bone marrow disease, develops from the neoplastic transformation of hematopoietic stem cells. Transformed hematopoietic stem cells give rise to abnormal white blood cells, also known as leukemia cells. Excessive production of leukemia cells in the body reduce the number of  healthy white blood cells, red blood cells, and platelets in blood and bone marrow resulting our body susceptible to any sort of infection, anemia, or easy bleeding. CML is a triphasic disease characterized by an initial chronic phase that is relatively benign and can last for several years. If untreated, CML progresses to an accelerated phase and/or blast phase, which is associated with increasing symptoms and worsening hematologic parameters. This disease mainly affects adults during or after middle age with a median age of diagnosis at around 65 years. In USA, the annual incidence rate of CML is approximately 4800 cases.

The chief molecular marker involved in the etiology of CML is the BCR-ABL fused gene. The BCR-ABL gene is formed by the fusion of tyrosine kinase gene ABL1 with the BCR gene through reciprocal translocation between chromosomes 9 and 22 during formation of the Philadelphia (Ph) chromosome. The Ph-chromosome is identified in over 95% of patients with CML and represents the genetic hallmark of CML.describe the image Several in vitro studies showed that the tyrosine kinase chimeric protein Bcr-Abl encoded by the BCR-ABL gene is constitutively active in leukemia cells and has oncogenic properties. Bcr-Abl chimeric protein has been found to be associated with genomic instability and thereby suggested to be responsible for progression to advanced phases of CML.

The discovery of the BCR-ABL gene and corresponding protein led to the synthesis of small-molecule drugs, aimed at inhibiting the tyrosine kinase activation of Bcr-Abl by competitive binding at the ATP-binding site. Imatinib mesylate (Gleevec) was the first tyrosine kinase inhibitor (TKI), approved by the FDA in 2001 for the treatment of chronic phase CML. Patients treated with imatinib exhibited hematological and cytogenetic responses with no disease progression to the advanced phase. However, a significant proportion of patients with CML did not achieve a satisfactory long-term response to imatinib treatment due to acquired resistance which is often caused by the mutation of the BCR-ABL gene. Both in vitro and in vivo studies discovered more than 90 mutations which are suggested to be associated with imatinib-resistance.  Among these mutations the “gate-keeper” mutation T315I appears to be the most common and present in up to 20% patients with CML. This mutation originated as a result of substitution of a threonine (T) residue with isoleucine (I) at amino acid position 315. Crystallographic analysis revealed that BCR-ABL gene mutations cause conformational changes in the ABL-kinase domain that interfere with imatinib binding, resulting in 30 to 40% resistance to imatinib.These findings led to the development of more potent 2nd generation TKIs – dasatinib (Sprycel) or nilotinib (Tasigna™). During clinical studies major cytogenetic response was observed in 35 to 63% of patients treated with dasatinib or nilotinib. In 2010 both TKIs received FDA approval for the treatment of CML patients who are resistant or intolerant to imatinib. Even though dasatinib and nilotinib showed efficacy against a number of imatinib-resistant mutants in CML, they are ineffective against subsets of mutants. In addition, imatinib, dasatinib, and nilotinib failed to show efficacy against the T315I mutant. Therefore, since until recently, the T315I mutation remained a clinical challenge in patients with primary or secondary resistance to dasatinib or nilotinib, whether their disease is newly diagnosed or imatinib-resistant.

A study published in The New England Journal of Medicine (November 29, 2012) by Cortes et al., reported the success of overcoming BCR-ABL T315I mutation mediated resistance to TKIs in CML with a new small-molecule TKI ponatinib (AP24534). In in vitro studies, potent activity of ponatinib was observed against all mutant forms of BCR-ABL (including T315I) at a concentration as low as 40 nM. In the phase I dose-escalation study of ponatinib, Cortes et al. observed complete cytogenetic response and major molecular response in CML patients with non-T315I mutations.describe the image Among chronic-phase CML patients with T315I mutation 100% exhibited hematologic response, 92% had a major cytogenetic response, 75% exhibited complete cytogenetic response, and 67% had a major molecular response. The most common side effects reported in the study include hypertension, rash, abdominal pain, fatigue, headache, dry skin, constipation, fever, joint pain, and nausea. Clinically promising similar results were also observed in the PACE trial, a multicenter, international, single-arm clinical trial of 449 patients with disease that was resistant or intolerant to prior tyrosine kinase inhibitor therapy. On December 14, 2012, the FDA approved ponatinib (Iclusig tablets) for the treatment of adult patients with all phases of CML that are resistant or intolerant to prior tyrosine kinase inhibitor therapy.



1.         O’Hare T, Deininger MW, Eide CA, et al. Targeting the BCR-ABL signaling pathway in therapy-resistant Philadelphia chromosome-positive leukemia. Clin Cancer Res. 2011;17:212-221.

2.         Cortes JE, Kantarjian H, Shah NP, et al. Ponatinib in refractory Philadelphia chromosome-positive leukemias. N Engl J Med. 2012;367:2075-2088.

3.         Huang X, Cortes J, Kantarjian H. Estimations of the increasing prevalence and plateau prevalence of chronic myeloid leukemia in the era of tyrosine kinase inhibitor therapy. Cancer. 2012;118:3123-3127.

4.         O’Hare T, Shakespeare WC, Zhu X, et al. AP24534, a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance. Cancer Cell. 2009;16:401-412.


The Shock of Sepsis: The Struggle to Treat SIRS

Since the mid-1880’s, scientists and clinicians have studied the concept of sepsis. Investigations started with physician-scientists such as Ignaz Semmelweiss, Joseph Lister, Hugo Schottmüller and Louis Pasteur, initiating the fields of immunology and diseases related to immunology that continue even to today. Unfortunately, while we increase our knowledge about sepsis (also known as systemic inflammatory response syndrome; SIRS), diagnosis and treatment remain difficult1,2. This is seen as recently as last year, when an infection of a 12-year-old boy , Rory Staunton, lead to fatal septic shock after doctors failed to see the early signs of SIRS3. His death initiated the formation of the Rory Staunton Foundation and subsequent reforms of emergency protocols taken in his home state of New York (labeled as “Rory’s Laws”)3.

describe the imageHowever, hospital policy reforms are just part of the issue; sepsis affects more than 700,000 people in North America every year, with a 30-50% mortality rate4. To date, there are no FDA-approved drugs to fight SIRS4. In fact, after 20 years of intense research into translational medicine for sepsis, none of the proposed treatment approaches have had enough clinical efficacy to be used as treatment paradigms1. Action must be taken to not only change the methodologies of care for sepsis, but to better understand the biological mechanisms of sepsis. From this understanding, we may be able to have more accurate detection of sepsis as well as more effective therapeutics.

Sepsis occurs when an infection causes a systemic inflammatory response5. This disease causes an imbalance of the immune system, leading to changes in the hemodynamics of the host, resulting in coagulation, heart ischemia, and multi-organ failure1.  Originally thought to be caused by overactivation of the innate immune system, many patients did not die from the initial onslaught of inflammation, but from later stages of immune system suppression (also known as “immunoparalysis”) which allowed opportunistic viruses and bacteria to take over the host1. The roles of sepsis may be analyzed by observing the different cell types it affects: the innate system, the adaptive immune system, and non-immune cells.

Since sepsis is due to bacterial and viral infection, the innate immune system is the most well studied aspect of the pathogenesis of sepsis. During SIRS the innate immune cells are pathologically affected: macrophages, dendritic cells and natural killer (NK) cells. During the initial period of infection, increased amounts of pathogen-associated molecular pattern (PAMPs) and damage-associate molecular pattern (DAMP) molecules are found in the host organism. Toll-like receptors (TLRs), such as TLR4 and TLR9, in the innate immune cells recognize these molecules and confer a host inflammatory response.  The inflammation leads to an increased presence of adhesion molecules on both innate and adaptive immune cells4 indicating the desire to enter the site of infection to confer an immune response.  Along the same lines, activation of the complement cascade occurs, leading to increased amounts of C5a protein. The upregulated C5a protein levels cause increased migration of innate immune cells and increased phagocytic ability of macrophages4.

Unfortunately, prolonged exposure of macrophages to C5a leads to dysregulation of macrophage activation and, eventually, apoptosis1. During this apoptotic stage, macrophages release high-mobility group protein B1 (HMGB1) which may lead to increased inflammation5. On the other hand, in neutrophils, sepsis is known to cause an abnormally long proliferation period5 which could lead to organ damage and increased inflammation. Surprisingly, sustained activation of neutrophils may also contribute to the immunoparalysis by sepsis because activated neutrophils increase reactive oxygen species, known to cause immunosuppression, and macrophages that eat apoptotic neutrophils express anti-inflammatory cytokines5. describe the imageProlonged sepsis also decreases the pro-inflammatory ability of dendritic cells; SIRS increases depletion of splenic and myeloid dendritic cells, where the remaining dendritic cells are functionally deficient4.  NK cells, originally shown to play a role in anti-viral immune responses but are also proposed to play a role in bacterial infection responses, have two distinct roles in different phases of sepsis.  In the early phases, NK cells are thought to contribute to the overactive immune responses and systemic inflammation4 however, in later phases of sepsis, NK cells may be compromised which could lead to secondary bacterial or viral infections, thus exacerbating the inflammatory response during SIRS4.

describe the imageIn addition to dysregulation of the innate immune system, the cells that make up the adaptive immune system, T-cells and B-cells, are also affected by sepsis. Sepsis decreases overall T-cell receptor function, and moves Th1 (proinflammatory) T-cells toward a Th2 (immunosuppressive) response1. In addition, CD25+Foxp3+ T-cells, also known as regulatory T-cells, are increased during SIRS5. Interferon-gamma, a proinflammatory cytokine expressed during sepsis, causes an increase in innate response activator (IRA) B Cells, B-cells that recognize PAMPs, impair infection clearance, and accelerate septic shock4,6. During sepsis, the increase of C5a causes T-Cell and B-cell apoptosis4, which leads to immunosuppression4.

Sepsis also has non-immunological effects on the patient. During sepsis, coagulation becomes more pronounced. This increase in coagulation and pre-coagulation states may lead to ischemic injury5.  Sepsis also causes cytopathic hypoxia5 and cardiomyopathy4. Interestingly, SIRS also plays a role on the autonomic nervous system, with increased apoptosis of the adrenal medullary cells, there is a dysregulation of the endocrine system that regulates the autonomic nervous system1.

Currently, there are several studies looking into the pathogenesis of sepsis with the goal being effective treatment. In terms of pathogenesis, recent studies looking at the role of STIM17  and PI3K activation in the initiation of sepsis5. Host deficiencies leading to sepsis are also being studied. Deficiencies, including,  zinc8 and ADAMS-T5 deficiencies, will hopefully elucidate more on the dysregulation found in sepsis. Currently, medical scientists are working with clinicians to study the more nuanced roles of hyper-responsiveness versus system immune suppression4. From these studies, multiple immunotherapies have been proposed: dendritic cell implantation4, regulatory T-cell implantation 4, and the modification of the host immune system by using TLR antagonists4, injections of the proinflammatory cytokines IL-15 and IL-174, and limiting adaptive immune system exhaustion by blocking PD-14. However, several of these studies, such as T-reg implantation, and using TLR antagonists have had limited clinical efficacy and have been prematurely terminated4. This may be due to the lack of an appropriate model to study human sepsis in the pre-clinical setting.

The limitations of current research methodologies for sepsis have shown the need for reform in SIRS research. Recently an article by Seok et al in the Proceedings of the National Academy of Sciences elucidated the limitations of using mouse models to studying human inflammation9. In this article, Seok et al list the differences in temporal responses, gene signatures, and regulated pathways involved inflammatory response following various insults9. The reasons for describe the imagedifferences in the inflammatory response may be described by the evolutionary differences between mouse and human immune systems, the inbred nature of the mice used, and the tendency to focus on a single mechanism in mouse models when there is great overlap of immune responses within a host system. These authors propose studying more of the genetic and epigenetic changes of patient samples during sepsis to find appropriate mouse models for study9. Furthermore, they propose in vitro recapitulation of the inflammation response in diseased tissue9. Unfortunately, the in vitro model is limited and this reductionist approach may miss a key cell, cellular event, or special/temporal arrangement that may be vital to pathogenesis of sepsis.

The use of non-human primates as a sepsis model may be more accurate than either of these, but the ethical and cost-effective issues of using non-human primates remains a deterrent. A final model that may be of use to sepsis researchers in the future is the use of the humanized mouse model; a severely immunocompromised mouse host that has had a recapitulation of the adaptive and/or innate immune systems for in vivo study of human pathologies.  The use of humanized mice for the study of sepsis was proposed by Unsinger et al, where they used two-day-old NOD-scid IL2 receptor-gamma knockout mice and transplanted them with hCD34+ enriched hematopoietic cord blood stem cells.  After establishment of the human immune system, mice were treated with cecal ligation puncture (CLP) model of intra-abdominal peritonitis and assayed for immune response changes10. These mice developed a functional human innate and adaptive immune system with recapitulation of the human immune response to sepsis.  However, while there still remain differences in the humanized mouse model versus the actual human immune response, such as differences in the bacterial flora that is found in the bowels10, this model nonetheless provides a useful tool which, along with more genetic and epigenetic information from patient studies, will lead to more impactful pre-clinical studies and possibly FDA-approved drugs to treat sepsis in the clinics.



1. Rittirsch, D., Flierl, M. A. & Ward, P. A. Harmful molecular mechanisms in sepsis. Nat Rev Immunol 8, 776-787, doi:10.1038/nri2402 (2008).

2. Stearns-Kurosawa, D. J., Osuchowski, M. F., Valentine, C., Kurosawa, S. & Remick, D. G. The pathogenesis of sepsis. Annu Rev Pathol 6, 19-48, doi:10.1146/annurev-pathol-011110-130327 (2011).

3. Dwyer, J. Death of a Boy Prompts New Medical Efforts Nationwide, October 26, 2012).

4. Ward, P. A. & Bosmann, M. A historical perspective on sepsis. Am J Pathol 181, 2-7, doi:10.1016/j.ajpath.2012.05.003 (2012).

5. Cinel, I. & Opal, S. M. Molecular biology of inflammation and sepsis: a primer. Crit Care Med 37, 291-304, doi:10.1097/CCM.0b013e31819267fb (2009).

6. Rauch, P. J. et al. Innate response activator B cells protect against microbial sepsis. Science 335, 597-601, doi:10.1126/science.1215173

7. Gandhirajan, R. K. et al. Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation. J Clin Invest, doi:10.1172/jci65647 (2013).

8. Liu, M. J. et al. ZIP8 Regulates Host Defense through Zinc-Mediated Inhibition of NF-κB. Cell Rep, doi:10.1016/j.celrep.2013.01.009 (2013).

9. Seok, J. et al. Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc Natl Acad Sci U S A, doi:10.1073/pnas.1222878110 (2013).

10. Unsinger, J., McDonough, J. S., Shultz, L. D., Ferguson, T. A. & Hotchkiss, R. S. Sepsis-induced human lymphocyte apoptosis and cytokine production in “humanized” mice. J Leukoc Biol 86, 219-227, doi:10.1189/jlb.1008615 (2009).

Maturing and Assaying Monocyte-Derived Dendritic Cells

Generating dendritic cells (DCs) from PBMC CD14+ monocytes allows researchers to do a host of immunological assays. A common example of this is to examine the reactivity of a T cell mixture to a certain antigen of interest. However, prior to doing such antigen presentation assays, DCs must be properly matured in order to fully elicit a T cell response.

After generating your monocyte derived DCs (mDCs) from PBMCs, as I described in my previous post, you will have several choices on how to mature them. Two of the most common choices are to either use LPS or a monocyte maturation cocktail (MMC). LPS binds TLR4, which results in a host of downstream inflammatory genes being upregulated. Addition of IFNγ can polarize the DCs to a Th1 phenotype, while the addition of TNFα can polarize the DCs somewhat towards a Th2 phenotype. MMC, however, usually involves the addition of several molecules including TNFα, IL-6, IL-1β, and PGE2. The overall effect of this pool of molecules is to elicit a mixed Th1 and Th2 response by the DCs. Thus, the maturation method of choice is a critical choice for the researcher and may vary depending on the downstream functional assay.

Interrogating your DCs by flow cytometry is a good idea so you can be sure you have attained the cell phenotype you desire. mDCs will commonly express CD11c and CD1c and should be CD123-. Furthermore, upregulation of costimulatory molecules CD80 and CD86 and the immunoregulatory molecule CD83 and downregulation of CD14 are hallmarks of DC maturation. HLA molecules are also significantly upregulated. Remember, these molecules are not just cell markers, but have important functional relevance. The upregulation of costimulatory molecules is critical for the activation of T cells and the upregulation of surface HLA molecules is a reflection is the enhance antigen presentation capability of a mature DC.Dendritic Cells Dot Plot with CD1c and CD11c Expression

Running your DCs on the flow cytometer will require a few special tweaks on your normal cytometer settings. The first thing you will notice is that the DCs are rather massive and irregular shaped cells. You will therefore likely need to significantly decrease both your forward scatter and side scatter to locate them on your dot plot. Secondly you will want to significantly decrease the voltages for all the channels detecting fluorchromes on your DC activation markers. These activation markers are expressed at such a high level on the DCs, that they are incredibly bright. A third issue is the high level of auto-fluorescence on DCs. It is always a good idea to have some extra DCs you can run while setting up your voltages to make sure your CD marker fluorochromes are all on scale.  Be sure to use the activated sample of DCs for this! Once you have verified your settings will work, you can then proceed to normal compensation set up.

Once your cytometer settings are established your cells are ready to assay. It is a good idea to have a sample of DCs that you did not stimulate as a control to compare your matured DCs to. In my experience the best way to compare markers, such as CD83, CD86, HLA-ABC, and HLA-DR, is by using histogram overlays. Their upregulation can often be a slight shift in fluorescent intensity, which you can readout by graphing Median Fluorescent Intensity (MFI). Of course be sure that you have titered your antibodies appropriately and use isotype controls when you can. Also keep in mind that comparing MFI readouts between different assay days, different stains, and different experiments is virtually impossible. Try to group your assays whenever possible, but if not, fold change in MFI is a useful, though not ideal, calculation for comparing these sorts of data.


Differentiation of Peripheral Blood Monocytes into Dendritic Cells. David W. O’Neill, Nina Bhardwaj. Current Protocols in Immunology. July, 2005.

Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood. Bender A, Sapp M, Schuler G, Steinman RM, Bhardwaj N.  J Immunol Methods. 1996 Sep 27;196(2):121-35.

Monocyte-derived DC maturation strategies and related pathways: a transcriptional view. Luciano Castiello, Marianna Sabatino,   Ping Jin, Carol Clayberger, Francesco M. Marincola, Alan M. Krensky, David F. Stroncek. Cancer Immunol Immunother. 2011 April; 60(4): 457–466.

Taking dendritic cells into medicine. Steinman RM, Banchereau J. Nature. 2007;449:419–426.

Current approaches in dendritic cell generation and future implications for cancer immunotherapy.  Tuyaerts S, Aerts JL, Corthals J, et al. Cancer Immunol Immunother. 2007;56:1513–1537.

Comparative evaluation of techniques for the manufacturing of dendritic cell-based cancer vaccines.  Dohnal AM, Graffi S, Witt V, et al. J Cell Mol Med. 2009;13:125–135. 

describe the imageColt Egelston is currently a post-doctoral fellow at the Beckman Research Institute of the City of Hope, in Duarte, CA. He received his Ph.D. from Rush University in Chicago and is interested in all things immunology.

Unique anti-tumor functions of IFNg vs. IL-17 producing CD8 cells

interferon gammaThe anti-tumor effectiveness of IFNγ-producing CD4+ TH1 cells and CD8+ TC1 cells is well accepted.  However, the role of IL-17 producing CD4+ (TH17) and CD8+ T cells (TC17) in promoting or inhibiting tumor growth remains unclear, as various studies have shown both tumor-inhibiting and tumor-promoting functions of these cell types.  Thus, context is a key determinant factor in the role of IL-17 producing T cell subsets in tumor immune responses.

In the current February 15, 2013 issue of The Journal of Immunology, Yu. et. al. compared the role of adoptively transferred tumor-specific TC1 and TC17 cells in controlling tumor growth.  In this murine model, anti-gp100 T cells from Pmel-1 TCR transgenic mice were polarized ex-vivo into TC1 or TC17 phenotypes, and adoptively transferred into luciferase-expressing B16F10 melanoma lung metastatic tumor-bearing mice that had undergone total body irradiation.

The efficacy of anti-tumor TC1 or TC17 cells in mediating tumor regression was monitored by measuring tumor burden by luciferase luminescence and overall survival.  By these measures, both TC1 and TC17 cells exhibited anti-tumor activity, however TC1 cells were superior.  TC1 cells completely inhibited tumor growth, while TC17 cells delayed tumor growth but were ultimately unable to control the tumor.

Adoptively transferred TC1 cells were found to produce only IFNγ but not IL-17, while TC17 cells expressed high levels of IL-17 and could also differentiate into IFNγ-producing cells, as is known for these cell subsets.  In a fascinating observation, the authors found that eliminating IFNγ-responsiveness in tumor cells completely reversed the relative efficacy of TC1 and TC17 cells. IFNγ-responsiveness in tumor cells was required for TC1 mediated anti-tumor activity, indicating a critical role for IFNγ-responsive genes in promoting tumor cell recognition by TC1 cells and/or growth inhibition or sensitivity to apoptosis. However, when tumor cells could no longer respond to IFNγ, TC17 cells were now able to induce complete tumor regression. In cytokine-neutralization assays, in vivo IFNγ was required however, for the anti-tumor effectiveness of TC17 cells while IL-17 was not.  This indicates that in the context of TC17 cell therapy, IFNγ is required for modulation of non-tumor cells in the tumor microenvironment, while IFNγ signaling in the tumor cells themselves is puzzlingly detrimental.

Within the first few days of adoptive transfer, TC1 cells proliferated faster in vivo than TC17 cells.  However, after two and four weeks, in vivo levels of TC17 cells were higher or similar in the spleen and lungs compared with TC1 cells in mice bearing wild-type as well as IFNγ-nonresponsive tumors.  Thus, the superior in vivo persistence of TC17 cells may be a factor in TC17 cell-elicited anti-tumor responses.  IFNγ elicits its effects through activation of the STAT1 transcription factor, while IL-17 signals through an alternate ACT1 pathway to activate NF-ĸBIL-22, also released by TC17 cells activates STAT3.  Thus an interaction between these pathways in tumor cells may mediate the differential requirement for IFNγ-responsiveness in TC1 vs. TC17 mediated anti-tumor effects.

While it remains unclear why TC17 cells in this model were able to effectively control IFNγ-nonresponsive tumor cells but not wild-type tumor cells, and TC1 cells exhibited the opposite propensity, these observations have important implications for future enactment of adoptive cell transfer for tumor therapeutics.  Because of their inherent stem-like propensity for long term in vivo persistence and demonstrations of highly effective anti-tumor functions, TH17 cells have been proposed to be a superior CD4+ cellular subset for adoptive anti-tumor T cell therapy in combinations with CD8+ T cells.  However, the observations in this study suggest that different cytokine-producing CD8+ TC and CD4+ TH subsets will vary in their effectiveness depending on factors such as the tumor’s ability to respond to cytokines including IFNγ.


Further Reading:

Adoptive Transfer of Tc1 or Tc17 Cells Elicits Antitumor Immunity against Established Melanoma through Distinct Mechanisms.  Yu Y, Cho HI, Wang D, Kaosaard K, Anasetti C, Celis E, Yu XZ. J Immunol. 2013 Feb 15;190(4):1873-81.

Tumor-specific Th17-polarized cells eradicate large established melanoma.  Muranski P, Boni A, Antony PA, Cassard L, Irvine KR, Kaiser A, Paulos CM, Palmer DC, Touloukian CE, Ptak K, Gattinoni L, Wrzesinski C, Hinrichs CS, Kerstann KW, Feigenbaum L, Chan CC, Restifo NP. Blood. 2008 Jul 15;112(2):362-73.

Phenotype, distribution, generation, and functional and clinical relevance of Th17 cells in the human tumor environments.  Kryczek I, Banerjee M, Cheng P, Vatan L, Szeliga W, Wei S, Huang E, Finlayson E, Simeone D, Welling TH, Chang A, Coukos G, Liu R, Zou W. Blood. 2009 Aug 6;114(6):1141-9.

Structure and signalling in the IL-17 receptor superfamily.  Sarah L. Gaffen.  Nat Rev Immunol. 2009 August; 9(8): 556.

Human TH17 cells are long-lived effector memory cells.  Kryczek I, Zhao E, Liu Y, Wang Y, Vatan L, Szeliga W, Moyer J, Klimczak A, Lange A, Zou W. Sci Transl Med. 2011 Oct 12;3(104):104ra100.

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