Melanoma is a type of skin cancer. It arises from specialized pigmented cells in our body known as melanocytes that are responsible for the production of melanin (a pigment responsible for skin and hair color). Because most melanoma cells still make melanin, melanoma tumors are usually brown or black. It accounts for 4% of all skin cancers; however, it is responsible for the largest numbers of skin cancer related death in the world. In the US, according to the national cancer institute, estimated new cases and deaths from melanoma in 2013 would be 76,690 and 9,480 respectively.

Several studies using molecular profiling and genomic sequencing have shown that melanoma is a disease of a heterogeneous group of tumors, and its progression is driven by specific oncogenic mutations. In 2002, Davies et al. first reported the presence of B-RAF somatic missense mutations in 66% of malignant melanomas. RAF (Rapidlydescribe the image growing Fibrosarcoma) protein is a serine/thereonine kinase. Three members of this kinase family are A-RAF, B-RAF, and C-RAF. These serine/threonine protein kinases, downstream of the membrane-bound small G protein RAS, are components of the mitogen activtated protein kinase (MAPK) signal transduction pathway. With closely overlapping functions, all members of the RAF family are associated with the activation of the MAPK pathway. Activation of the MAPK pathway has been associated with uncontrolled growth and drug resistance in several tumors. Researchers have identified over 50 distinct mutations in the B-RAF gene so far. However, most of these mutations are extremely rare. The most common mutation in melanoma, accounting for 90% of all B-RAF mutations, is the V600E mutation that occurs as a result of substitution of amino acid valine (V) to glutamic acid (E) at codon 600. Approximately 50% of melanomas harbor the V600E B-RAF mutation, while other mutations observed in melanomas are usually associated with the activation of N-RAS and c-KIT.

Several studies reported association of the V600E B-RAF mutation with the progression of melanoma. In a pre-clinical study Smalley et al. (2010) observed tumor formation in immunocompromised mice following introduction of mutant B-RAF in melanocytes. Inversely, in their study, Smalley et al. also observed that inhibition of mutated B-RAF using RNA-interference resulted in tumor cell death. In addition, several other studies reported that inhibition of V600E mutant B-RAF prevents melanoma cell proliferation, induces apoptosis (programmed cell death), and also blocks melanoma xenograft growth in vivo. Even though many studies suggested that V600E B-RAF mutation may not be sufficient alone for melanoma induction, a wealth of evidence demonstrated that mutated B-RAF is necessary for the maintenance and progression of melanoma in human. Therefore, mutated B-RAF represents a therapeutic target in melanoma, which is why several B-RAF kinase inhibitors have already been developed. Sorafenib was the first B-RAF inhibitor studied in melanoma patients. In addition, vemurafenib (Zelboraf) and dabrafenib (GSK2118436) were also studied in melanoma patients with V600E B-RAF mutations.  In 2011 vemurafenib received FDA approval for the treatment of melanoma patients harboring the V600E B-RAF mutation. In clinical trials, in which patients were undergoing treatment with vemurafenib, the drug reduced risk of death by 63% and risk of progression by 74%.

At present several clinical trials also evaluate clinical efficacy of vemurafenib in combination with leflunomide  (antirheumatic drug), GDC-0973 (MEK inhibitor), and metformin (antidiabetic drug). In addition, several other drugs targeting B-RAF and its downstream pathway are also in development. Therefore, further improvements can be expected in this personalized and targeted therapy in melanoma.



1. Ascierto, P. A., Kirkwood, J. M., Grob, J. J., Simeone, E., Grimaldi, A. M., Maio, M., Palmieri, G., Testori, A., Marincola, F. M., and Mozzillo, N. (2012). The role of BRAF V600 mutation in melanoma. J Transl Med 10, 85.

2.Davies, H., Bignell, G. R., Cox, C., Stephens, P., Edkins, S., Clegg, S., Teague, J., Woffendin, H., Garnett, M. J., Bottomley, W., et al. (2002). Mutations of the BRAF gene in human cancer. Nature 417, 949-954.

3. Smalley, K. S. (2010). Understanding melanoma signaling networks as the basis for molecular targeted therapy. J Invest Dermatol 130, 28-37.

The Immunoscore: bringing immunological parameters to the clinic for cancer patient prognosis

Cancer stagesClassical prognosis of cancer patients utilizes the AJCC/UICC (American Joint Committee on Cancer / International Union Against Cancer) “TNM” classification system, in which T (Tumor) is indicative of primary tumor size and invasion properties, N (Nodes) indicates the extent of tumor invasion into draining and regional lymph nodes, and M (Metastasis), describes the presence and extent of metastatic lesions at diagnosis.  The combinations of these parameters are then used to assess a patient’s stage at diagnosis and predict patient outcome.  The exact parameter definitions vary for each cancer type.

However, it has long been known that while the TNM system provides a fairly good estimate for patient populations overall, there is still significant heterogeneity within each stage as to tumor recurrence and ultimate outcome.  This is unsurprising given this staging system evaluates only tumor characteristics and fails to account for other patient parameters. In particular, the integrity of the patient’s immune system, our inherent natural protection against tumor development, has been shown to have a significant impact on disease progression and patient outcome.

In colorectal cancer as well as other cancer types, much progress has been made in ascertaining the prognostic significance of cytotoxic CD8+ T cell infiltration into the tumor microenvironment.  The densities of CD8+ T cell presence in the invasive margin (IM) and the center of the tumor (CT) have been shown to have significant prognostic value.  In a study by Pages et. al. (J Clin Oncol. 2009), assessment of CD8+ T cells and CD45RO+ memory T cell densities in CT/IM tumor regions in stage I and II colorectal cancer patients significantly predicted recurrence and overall survival, showing that application of this system is particularly relevant in early stage patients to better direct treatment strategies.  Multiple cytotoxic CD8+ T cell and TH1 phenotyping markers have shown prognostic significance in human cancer patients, including CD8, CD3, CD45RO, and Granzyme B expression.  However, as discussed by Dr. Jerome Galon in the Oct 3, 2012 J Transl Med. article, CD8 and CD3 represent the most robust markers for adoption into routine clinical practice as CD45RO, and Granzyme B expression are intensity-dependant evaluations and thus much more technically difficult to standardize.

Thus, the proposed immunoscore relies on immunohistochemistry staining for CD8+ and CD3+ T cells in CT/IM tumor regions using standardized antibodies and protocols.  Quantitative assessment of their densities is then determined and scored on whole tissue slides using specified slide scanning and staining analysis software.

In 2012, an international task force, led by Dr. Galon was established to promote the routine usage of this classification system in clinical diagnosis of cancer patients.  The goals of this taskforce include feasibility and standardization of the quantitative immunohistochemistry protocol used to derive the score, worldwide validation of the immunoscore for colorectal cancer patient prognosis, as well as the application of this classification system for other cancer types.  Thus, the immunoscore may soon become a standard clinical practice and aid in better prognostic stratification of patients and therapeutic guidance.


Further Reading:

Website: American Joint Committee on Cancer Staging

Cancer classification using the Immunoscore: a worldwide task forceGalon J, Pagès F, Marincola FM, Angell HK, Thurin M, Lugli A, Zlobec I, Berger A, Bifulco C, Botti G, Tatangelo F, Britten CM, Kreiter S, Chouchane L, Delrio P, Arndt H, Asslaber M, Maio M, Masucci GV, Mihm M, Vidal-Vanaclocha F, Allison JP, Gnjatic S, Hakansson L, Huber C, Singh-Jasuja H, Ottensmeier C, Zwierzina H, Laghi L, Grizzi F, Ohashi PS, Shaw PA, Clarke BA, Wouters BG, Kawakami Y, Hazama S, Okuno K, Wang E, O’Donnell-Tormey J, Lagorce C, Pawelec G, Nishimura MI, Hawkins R, Lapointe R, Lundqvist A, Khleif SN, Ogino S, Gibbs P, Waring P, Sato N, Torigoe T, Itoh K, Patel PS, Shukla SN, Palmqvist R, Nagtegaal ID, Wang Y, D’Arrigo C, Kopetz S, Sinicrope FA, Trinchieri G, Gajewski TF, Ascierto PA, Fox BA.  J Transl Med. 2012 Oct 3;10:205.

The immune score as a new possible approach for the classification of cancer.  Galon J, Pagès F, Marincola FM, Thurin M, Trinchieri G, Fox BA, Gajewski TF, Ascierto PA. J Transl Med. 2012 Jan 3;10:1.

The immune contexture in human tumours: impact on clinical outcome. Fridman WH, Pages F, Sautes-Fridman C, Galon J.  Nat Rev Cancer 2012, 12:298-306.

In situ cytotoxic and memory T cells predict outcome in patients with early-stage colorectal cancerPagès F, Kirilovsky A, Mlecnik B, Asslaber M, Tosolini M, Bindea G, Lagorce C, Wind P, Marliot F, Bruneval P, Zatloukal K, Trajanoski Z, Berger A, Fridman WH, Galon J. J Clin Oncol. 2009 Dec 10;27(35):5944-51.

Image courtesy of simplyanon on Wikipedia

Does Salt Contribute to Autoimmunity?

Several recent findings have identified variants in predominantly immune-response genes as underlying contributors in autoimmune diseases’ development. Nonetheless, along with other autoimmune disorders, there has been a significant increase particularly in incidences of multiple sclerosis (MS) and type-1 diabetes. These observations imply that in addition to the genetic causality, specific changes in environmental factors are significant contributors to the recent increases in autoimmune disorders.

Maintenance of immune system’s homeostasis is dependent on highly specified regulation of pro-inflammatory and regulatory CD4+ helper T-cell populations. TH17 cells are pathogenic pro-inflammatory T-cells that produce a proinflammatory cytokine, Interleukin 17A (IL-17) and while they play a major role in immune defense against extracellular pathogens, they also play a major role in induction of autoimmune disorders, such as MS, psoriasis, rheumatoid arthritis, and type-1 diabetes, for which T-cell modulation is a common treatment regime.

CD4 T-cells,TH17,MS,autoimmune

Three previously identified factors, Interleukin 6 (IL-6), Transforming growth factor β (TGFβ) and Interleukin-23 subunit alpha (IL-23) are key players in the induction of naïve CD4+ T-cells to differentiate into TH17 cells. However, the complete mechanism and factors responsible for stimulating naïve CD4+ T-cells’ differentiation into TH17 cells is not well understood. Understanding of these underlying factors is crucial for developing therapeutic strategies to control TH17 cell differentiation.

Three complementary, collaborative studies published in Nature this week report compelling insights on the regulatory mechanism of TH17 cell differentiation and subsequent inflammatory response, while revealing evidence of interplay of genetics and a new environmental factor involved in autoimmune disease susceptibility.

By utilizing a unique approach, consisting of combination of transcriptional profiling, novel computational methods and nanowire-based short interfering RNA (siRNA) delivery to construct, Regev’s group was able to and identify 12 novel regulators and decipher the complex transcriptional network of TH17 differentiation; they found that TH17 differentiation is regulated by two intra-connected, but antagonistic networks, such that one module promotes TH17 differentiation and proliferation while suppressing the development of other T-cells, whereas the antagonist module suppresses TH17 cells.

By upregulating the expression of IL-23 receptor (IL-23R) on TH17 cells, IL-23 sustains the TH17 inflammatory response and induces its pathogenic effector functions. Through transcriptional profiling, Kuchroo’s group found that a serine/threonine kinase,  Serine/threonine-protein kinase 1 (SGK1), an essential node downstream of IL-23 signaling, is crucial for regulating IL-23R expression and stabilizing the TH17 cell pathogenic effector function by inhibiting the molecule that represses IL-23R expression.

Previous studies have shown that SGK1 regulates Na+ transport and NaCl (salt) homeostasis in cells. Interestingly, Kuchroo’s study shows the positive correlation between increased concentration in salt and SGK1 expression, followed by upregulation of IL-23R and enhanced TH17 differentiation in vitro as well as in vivo.

Consistent with Kuchroo’s finding, Hafler’s group reported that excess NaCl uptake can affect the innate immune system, and provided compelling data suggesting the possibility of the direct positive correlation between increased salt intake and incidence of autoimmune disease.

Previous studies have suggested the involvement of environmental factors, such as viruses, smoking, lack of sunlight and Vitamin D in different autoimmune disorders.  Salt, as the new potential environmental factor in autoimmune diseases is certainly an intriguing notion; not only due to the relevant molecular mechanisms reported in the three new studies, but also due to the fact that in most developed countries, the consumption of processed foods (such as fast foods), which contain over 100 times more NaCl compared to non-processed meals, has increased significantly over the past three decades. Nonetheless, future epidemiological studies in humans are needed to further investigate the possible correlation between salt consumption and autoimmunity incidence.

Further Readings:

Induction of Pathogenic TH17 Cells by Inducible Salt-sensing Kinase SGK1

Dynamic Regulatory Network Controlling TH17 Cell Differentiation

Sodium Chloride Drives Autoimmune Disease by The Induction Of Pathogenic TH17 Cells


Breast cancer is the most common cancer in women and the second-leading cause of cancer-related death in women worldwide. Despite progresses in the treatment of early stage breast cancer, approximately one third of patients will develop metastatic breast cancer (MBC). According to the National Cancer Institute, in USA, the estimated new cases and deaths from breast cancer in 2013 would be 232,340 and 39,620 respectively.

Approximately 20%–30% of breast cancers exhibit increased expression of human epidermal growth factor receptor 2 (HER-2/neu) caused by amplification of the erb-B2 oncogene. Breast cancers with elevated HER-2 expression are known as HER2-positive cancers. HER-2-positive breast cancers are more aggressive than other breast cancers. Patients with these tumors have a poorer prognosis and decreased chance of survival compared with patients whose tumors do not overexpress HER-2.

describe the imageHER-2 is a 185-kDa orphan transmembrane receptor tyrosine kinase. Dimerization of HER-2 with ligand- bound HER-3 or HER-4 receptor activates signaling pathways inside the cell. Activated HER-2 signaling stimulates cell proliferation and survival via activation of the MAPK and PI3K/Akt/mTOR pathways. Collectively these signaling pathways result in uncontrolled growth of the tumor. Several studies suggested that the overexpression/amplification of HER-2 may lead to the development and progression of pre-malignant breast disease and also tumor metastasis. Therefore, the association of HER-2 in breast cancer as well as its involvement in tumor aggressiveness makes this receptor an appropriate target for tumor-specific therapies. Several strategies have been developed to inhibit HER-2 signaling. These include a tyrosine kinase inhibitor called lapatinib and a recombinant humanized monoclonal antibody called trastuzumab (Herceptin®).  In this post I will focus only on trastuzumab mediated therapy in breast cancer.  Trastuzumab binds to the extracellular domain of the HER-2 receptor. This inhibits HER-2 signaling via MAPK and PI3K/Akt cascades. In addition, trastuzumab binding also increases membrane localization of the tumor suppressor gene phosphatase and tensin homolog (PTEN), and inhibitor of the PI3K/Aktpathway.

In 1998 trastuzumab was approved for thetreatment of metastatic breast cancer (MBC), and in 2006 for the adjuvant treatment of HER2-overexpressing breast cancer. In early-stage breast cancer, treatment with trastuzumab and a neoadjuvant chemotherapy substantially improves overall survival (OS) and reduces the risk of recurrence, both by 33%. In MBC, trastuzumab treatment in combination with chemotherapy increases the time to progression of the disease by 49% and improves OS by 20%.

However, even though trastuzumab treatment substantially improves outcomes in both early-stage and MBC, both de novo and acquired resistance after initial response was observed. It is suggested that most patients with HER2-positive MBC will eventually develop resistance and have disease progression following trastuzumab treatment.

Several studies reported involvement of multiple factors in resistance to HER2-targeted therapy. These include hindrance to HER-2-trastuzumab binding, signaling through alternative pathways (for e.g. insulin-like growth factor receptor 1, vascular endothelial growth factor receptor) upregulation of signaling pathways downstream of HER-2, increased expression of heat shock protein 90 (HSP90), loss of PTEN and thereby constitutive activation of the PI3K/Akt pathway, and failure to induce an appropriate immune response.

To overcome transtuzumab-resistance, various treatment strategies have been developed. One strategy involves continuation of transtuzumab treatment in combination with a chemotherapeutic agent. In multiple pre-clinical and clinical studies, combination of T DM1 binding resized 600trastuzumab with taxanes docetaxel (Taxotere®) and paclitaxel (Taxol®) exhibited promising response in HER-2–overexpressing metastatic breast cancer.

A new strategy to increase efficacy of trastuzumab has also been developed using antibody-drug conjugate (ADC) technology. The antibody-drug conjugate trantuzumab emtansine (T-DM1, Kadcyla) is consist of trastuzumab bound to maytansinoid (or DM1, a potent microtubule inhibitor) through a nonreducible thioether linkage. T-DM1 binds to HER-2 positive tumor cells and thought to inhibit HER-2 signaling. This ADC also induces body’s immune response to attack cancer cells. Once inside the tumor cells, T-DM1 is designed to kill tumor cells by releasing DM1 which is a potent inhibitor of microtubule assembly, thereby causing cell death inside the cells.

In in vitro and preclinical studies T-DM1 inhibited growth of breast cancer cells which are cross-resistant to trastuzumab. T-DM1 was found well tolerated in phase I clinical study of breast cancer patients who had disease progression with earlier trastuzumab based treatment. In phase II study, increased progression-free survival (PFS) was observed in patients treated with T-DM1 compared to trastuzumab plus doecetaxel treatment. A clinical study published by Verma et al. (2012) reported that T-DM1 significantly prolonged PFS and OS in patients with HER-2 positive MBC previously treated with trastuzumab and a taxane. The most common side effects of T-DM1 treatment include low platelet count, low RBC count, nerve problems, and tiredness. On the basis of clinical efficacy of T-DM1 observed in phase I and II trials, a multicenter phase III trial (also known as EMILIA trial) was performed. This trial also observed increased PFS, reduction of risk of death, and fewer adverse events in T-DM1 treated patients compared to capecitabine plus lapatinib treatment (another first-line treatment option for HER-2positive MBC).

On February 22nd, 2013, the US food and drug administration (FDA) approved T-DM1 (Kadcyla) for the treatment of HER-2 positive MBC that has progressed following treatment with trastuzumab and a taxane.


Suggested reading:

[1] M.F. Barginear, V. John, D.R. Budman, Trastuzumab-DM1: A Clinical Update of the Novel Antibody-Drug Conjugate for HER2-Overexpressing Breast Cancer, Mol Med, 18 (2013) 1473-1479.

[2] M. Barok, M. Tanner, K. Köninki, J. Isola, Trastuzumab-DM1 causes tumour growth inhibition by mitotic catastrophe in trastuzumab-resistant breast cancer cells in vivo, Breast Cancer Res, 13 (2011) R46.

[3] M.S. Mohd Sharial, J. Crown, B.T. Hennessy, Overcoming resistance and restoring sensitivity to HER2-targeted therapies in breast cancer, Ann Oncol, 23 (2012) 3007-3016.

[4] S. Verma, D. Miles, L. Gianni, I.E. Krop, M. Welslau, J. Baselga, M. Pegram, D.Y. Oh, V. Diéras, E. Guardino, L. Fang, M.W. Lu, S. Olsen, K. Blackwell, E.S. Group, Trastuzumab emtansine for HER2-positive advanced breast cancer, N Engl J Med, 367 (2012) 1783-1791.


How do HSCs deal with aging?

hematopoietic stem cells resized 600Mature blood cells are relatively short-lived, and require replenishment from multipotent HSCs. Thus, HSCs must self-renew to generate an adequate pool of HSCs, as well as differentiate to give rise to more mature blood cells.  A balance between self-renewal and differentiation ensures that the hematopoietic system can be functionally sustained throughout the lifetime of an adult body.  However, as HSCs age, they accumulate DNA damage, often compromising their functionality.  DNA damage can be further propagated both to daughter stem cells and downstream lineages, and may increase the risk of developing blood disorders 1.

Depending on the nature of the damage, cells use two major response pathways to combat cellular stress.  If the damage is excessive and functionality is compromised, cells usually undergo apoptosis for self-elimination.  In contrast, autophagy allows cells a window of survival.  Autophagy is a process of self-degradation in which organelles or portions of the cytoplasm are sequestered within double-membrane vesicles, known as autophagosomes, and then delivered to lysosomes for degradation 2.  The resulting breakdown products are released through permeases and recycled in the cytosol.  Thus, autophagy can be used to generate high-energy compounds during conditions of metabolic stress.

Recently, in Nature, Warr et al found that metabolic stress and old age induce autophagy in HSCs 3.  The authors isolated HSCs and myeloid progenitors from the bone marrow of transgenic mice systemically expressing GFP fused to LC3, an autophagosome marker 4.  They used cytokine withdrawal to induce metabolic stress and measured autophagy induction by examining the formation and turnover of LC3-GFP.  Myeloid progenitors expressed LC3-GFP in the presence and absence of cytokines.  In contrast, HSCs did not express LC3-GFP in the presence of cytokines, but demonstrated autophagosome formation following cytokine withdrawal.  Furthermore, when the mice were starved in vivo, autophagy flux increased in HSCs.

The authors speculated that autophagy “protects” HSCs from starvation-induced apoptosis, and indeed, hematopoietic-specific deletion of an essential autophagy machinery component, ATG12, resulted in a significant increase in caspase activation in starved HSCs in vivo.  FOXO3A was identified as the specific transcriptional regulator that maintains pro-autophagy gene expression, and was expressed higher in HSCs compared to progenitors.  Interestingly, HSCs isolated from old mice retained their autophagic potential, and was found to be required for their survival.

In summary, Warr et al demonstrated that long-lived HSCs mount a protective survival autophagy response to combat metabolic stress, whereas short-lived progenitors do not.  Previous studies suggested that impaired autophagy might contribute to the aging phenotype 5.  However, this study directly showed that the pro-autophagy gene expression program is still intact in old HSCs and is essential for continued survival of these cells.  Future studies will address whether autophagy increases the incidence of age-related blood disorders since it protects damaged, old HSCs from elimination by apoptosis.



1          Rossi, D. J., Jamieson, C. H. & Weissman, I. L. Stems cells and the pathways to aging and cancer. Cell 132, 681-696, doi:10.1016/j.cell.2008.01.036 (2008).

2          He, C. & Klionsky, D. J. Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 43, 67-93, doi:10.1146/annurev-genet-102808-114910 (2009).

3          Warr, M. R. et al. FOXO3A directs a protective autophagy program in haematopoietic stem cells. Nature 494, 323-327, doi:10.1038/nature11895 (2013).

4          Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T. & Ohsumi, Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell 15, 1101-1111, doi:10.1091/mbc.E03-09-0704 (2004).

5          Rubinsztein, D. C., Marino, G. & Kroemer, G. Autophagy and aging. Cell 146, 682-695, doi:10.1016/j.cell.2011.07.030 (2011).


Generation of CD4+ Th1 cells from human PBMC

CD4+ T helper type 1 (TH1) cells are the effector T cell population that governs cell mediated immune responses against intracellular pathogens including viruses and intracellular bacteria.  TH1 cells mediate their effect by secreting cytokines such as interferon-gamma (IFNγ) and IL-2, and express cell surface markers including CXCR3 and CCR5 and the characteristic TH1 master transcription factor T-bet (TBX21) which can also be used for detection of TH1cells by flow cytometry, as discussed in a previous blog post.

Differentiation of naïve human CD4+ T cells down the TH1 pathway involves cytokines such as IL-12 which activates STAT4, and induces expression of IFNγ and T-bet.  As such, in vitro protocols differentiating peripheral blood mononuclear cells (PBMC)-derived naïve CD4+ T cells into TH1 cells involves incubation with IL-12 in the context of T cell activation through the T cell receptor (TCR) complex.

In my experience, TH1 cells are by far the easiest CD4+ helper T cell population to generate in vitro.  In order to generate TH1 cells from human PBMC, naïve CD4+ T cells must first be isolated.  Multiple methods of naïve CD4+ T cell isolation can be utilized, and magnetic bead- based methods are common and easy methods.  Companies such as Miltenyi Biotech and Stem Cell Technologies offer kits for isolation of untouched naïve CD4+ T cells from PBMC by negative isolation methodologies.

Following isolation, naïve CD4+ T cells are activated through the TCR complex.  Tissue culture plates can be coated with anti-CD3 (OKT1) and anti-CD28 antibodies in PBS prior to culture.  Alternatively, naïve CD4+ T cells can be cultured with Dynal CD3/CD28 T Cell Expander Dynabeads (Life Technologies) at a 1 bead per cell ratio.  A third alternative involves coating tissue culture plates with anti-CD3 alone and obtaining CD28 co-stimulation by the addition of autologous monocytes isolated from PBMCs into the culture.

To generate TH1 cells, recombinant human IL-12 is added alone, or at a lower dose in combination with anti-IL-4 blocking antibodies to inhibit the counteractive effects of IL-4 and TH2 pathways on TH1 cell polarization.  Finally recombinant human IL-2 is added to promote T cell proliferation.  Media and cytokines/blocking antibodies are refreshed every two to three days depending on the cell density, and as the cells expand the time to refresh the media shortens.

Lymphocyte activationTH1 cells can be generated and assayed for functions including IFNγ expression in as few as three days.  If long term or clonal T cells assays are of interest, cells can be expanded in the presence of IL-2 for 2-3 weeks following single cell cloning.  As previously discussed, TH1 cells can be identified by IFNγ expression following a 4-6 hour incubation with TCR activation by plate bound anti-CD3 plus anti-CD28, CD3/CD28 Dynabeads, or PMA/ionomycin in the presence of brefeldin-A.  Cells are then fixed, permeabilized, and stained for cell surface markers and intracellular IFNγ.

Finally, as a comparison, tandem experiments can be run in which naïve CD4+ T cells are maintained under non-polarizing (TH0) conditions.  For this, often no cytokines aside from IL-2 are added.  However the addition of anti-IL-12 and anti-IL-4 may be necessary to inhibit any cells from differentiating down TH1 or TH2 pathways by production of these cytokines by the T cells themselves.

In conclusion, generation of CD4+ TH1cells from human PBMC is a relatively simple and straightforward protocol, and very high percentages of TH1cells can be obtained through optimized protocols.


Further Reading:

Differentiation of effector CD4 T cell populations (*).  Zhu J, Yamane H, Paul WE.  Annu Rev Immunol. 2010;28:445-89.

Memory and flexibility of cytokine gene expression as separable properties of human T(H)1 and T(H)2 lymphocytes.  Messi M, Giacchetto I, Nagata K, Lanzavecchia A, Natoli G, Sallusto F.  Nat Immunol. 2003 Jan;4(1):78-86.

A critical function for transforming growth factor-beta, interleukin 23 and proinflammatory cytokines in driving and modulating human T(H)-17 responses.  Volpe E, Servant N, Zollinger R, Bogiatzi SI, Hupé P, Barillot E, Soumelis V. Nat Immunol. 2008 Jun;9(6):650-7.

Generation of Red Blood Cells from Human Pluripotent Stem Cells

After the brief review of the in vitro systems for hematopoietic differentiation of pluripotent stem cells (PSCs), I would now like to take a closer look at the functional properties of PSCs-derived blood cells and discuss their potential for clinical application.

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Erythrocytes or red blood cells (RBCs) are the most abundant cell population comprising of ~45% of the total blood volume, whose main function is to deliver oxygen to the body tissues. Red blood cells lack a nucleus and most organelles to provide maximum space for hemoglobin – a complex metalloprotein containing heme groups whose iron atoms temporarily bind to oxygen molecules and release them throughout the body.

Mammalian erythroid progenitors originate from a megakaryocyte-erythroid progenitor (MEP) and undergo the gradual process toward terminal differentiation. Two globin gene switches occur during development: the embryonic to fetal globin switch, which coincides with the transition from embryonic (yolk sac) to definitive (fetal liver) hematopoiesis; and fetal to adult switch, which occurs during the perinatal period. During erythroblast differentiation, the chromatin condenses while the hemoglobin concentration increases. Chromatin condensation involves histone deacetylation and unknown signals that activate the Rac-GTPases-mDia2 pathway, which is required for the formation of a contractile actin ring and subsequent enucleation, the process in which the nucleus is rapidly squeezed out of the cell.

In vivo, erythroid precursors proliferate, differentiate, and enucleate within specialized niches called erythroblastic islands. These hematopoietic compartments are composed of erythroblasts surrounding a central macrophage. The central macrophage communicates with erythroblasts through a number of signaling molecules and phagocytizes their nuclei after enucleation.


In 1977, the American biochemist Eugene Goldwasser isolated the human protein erythropoietin (EPO), which stimulates red blood cell production. EPO became a blockbuster product that changed the lives of millions of patients suffering from anemia. Although EPO with other specific additives allow red blood cells to mature in vitro without a supportive role of macrophages, the resulting proliferation and enucleation efficiency of red blood cells is lower than their capacities in vivo, suggesting the importance of a niche microenvironment.

Blood transfusions are a common treatment for severe anemia and massive blood loss due to trauma. A type O-negative red blood cell can be transfused to patients of all blood types and is always in great demand. Thus, the derivation of (O)Rh-negative RBCs from PSCs could be an effective way to overcome shortages in donated red blood cells.

Red blood cells can be produced from human pluripotent stem cells (hESCs and hiPSCs) through various differentiation systems, such as an embryoid body (EB) formation and coculturing hPSCs on top of stromal feeder cells. In general, the existing methods are sufficient for a large-scale production of hPSC-derived red blood cells, whose in vitro expansion capacity is greater than the expansion potential of the bone marrow, peripheral blood, or even cord blood-derived erythroid progenitors. Despite the large amounts of RBCs obtained in many studies, the majority of the resulting RBCs expresses embryonic ε– and fetal γ-globins with low levels of detectable adult β-globin. Although no differences were observed between hiPSC and hESC lines in terms of erythroid commitment and expression of erythroid markers, iPSC-derived red blood cells have lower proliferation activity and produce less enucleated cells.

Robert Lanza’s group suggested the idea of developing an early hemato-endothelial progenitor, a hemangioblast, which can be expanded and cryopreserved.This study, published in Nature Methods in 2007, demonstrated the regenerative properties of blast cells that differentiate into multiple hematopoietic lineages as well as into endothelial cells. The extended coculture of these cells on OP9 feeders facilitated enucleation in up to 65% of cells and the expression of β-globin in up to 15% of the cells.

Lapillonne and colleagues employed a feeder free, two-step differentiating system to produce mature blood cells from hESCs and  iPSCs. In the first step, researchers initiated erythropoiesis by conditioning embryoid bodies in the presence of cytokines. To obtain mature erythrocytes, they further cultured cells in the presence of EPO, SCF, IL3 and 10% of human plasma for another 25 days. The resulting population contained up to 10% of enucleated cultured RBC from hiPSC, and 66% of enucleated RBC from hESC. The vast majority (~93%) of PSCs-derived red blood cells expressed the tetrameric form of fetal hemoglobin HbF (α2γ2). The CO-rebinding kinetics of hemoglobin from hESC- and hiPSC-derived erythroid cells was almost identical to those of cord blood cells suggesting that the HbF in these erythrocytes is functional.

Several studies have shown a time-dependent increase in β-globin expression, the oxygen dissociation curve and G6PD activities similar to normal RBCs. Nevertheless, significant progress is needed in the production of terminally differentiated/enucleated erythrocytes. Thus, at least two major steps are required for future therapeutic use of in vitro generated RBCs: (i) finding a cost-effective method for generating fully maturated, enucleated erythrocytes, and (ii) the evaluation of their biophysical parameters such as membrane surface potential, pliability, half-life in vivo, hemoglobin packing, gas exchange properties, and immunogenicity.


Further Reading:

1. Peng Ji, Maki Murata-Hori, Harvey F. Lodish Formation of mammalian erythrocytes: Chromatin condensation and enucleation Trends Cell Biol. 2011 July; 21(7): 409–415.

2. Joel Anne Chasis, Narla Mohandas Erythroblastic islands: niches for erythropoiesis Blood. 2008 August 1; 112(3): 470–478.

3. Lu SJ, Feng Q, Caballero S, Chen Y, Moore MA, Grant MB, Lanza R. Generation of functional hemangioblasts from human embryonic stem cells. Nat Methods. 2007 Jun;4(6):501-9.

4. Hélène Lapillonne, Ladan Kobari, Christelle Mazurier et al. Red blood cell generation from human induced pluripotent stem cells: perspectives for transfusion medicine Haematologica. 2010 October; 95(10): 1651–1659.

5. Dias J, Gumenyuk M, Kang H, Vodyanik M, Yu J, Thomson JA, Slukvin II. Generation of red blood cells from human induced pluripotent stem cells. Stem Cells Dev. 2011 Sep;20(9):1639-47.

6. Chang KH, Bonig H, Papayannopoulou T. Generation and characterization of erythroid cells from human embryonic stem cells and induced pluripotent stem cells: an overview. Stem Cells Int. 2011;2011:791604.



1. Scanning electron microscope (SEM) image of a single red blood cell on the tip of a needle:

2. Confocal immunofluorescence image of an island reconstituted from freshly harvested mouse bone marrow cells stained with erythroid-specific marker (red), macrophage marker (green) and DNA probe (blue). Central macrophage is indicated by an arrow and a multilobulated reticulocyte by an arrowhead. Joel Anne Chasis, Narla Mohandas Erythroblastic islands: niches for erythropoiesis Blood. 2008 August 1; 112(3): 470–478.

Induction of Tumor cell senescence by TH1 cytokines IFN-g and TNF

interferon betaInterferons, including type I (IFNα/β) and type II (IFNγ) are known to be critical for mediating multiple aspects of tumor immunity, by targeting both immune cells for activation, and cancer cells for expression of MHC and genes associated with growth arrest and apoptosis.  Thus, expression of cytokines such as IFNγ by immune cells has been shown to be critical in anti-tumor immune responses.  IFNγ is one of the major effector cytokines of CD4+ TH1 cells and cytotoxic CD8+ T cells.  However, the full effects of IFNγ as well as other TH1 cytokines in mediating anti-tumor effects have not been fully elucidated.

Previous observations were made that tumor-specific CD4+ TH1 cells, IFNγ, and TNF were required for controlling tumor growth.  However, tumor growth arrest induced by IFNγ and TNF occurred without either significant T cell infiltration into the tumor, or appreciable tumor cell destruction (Müller-Hermelink et. al, 2008).  Thus, the anti-tumor mechanisms of the combined action of IFNγ and TNF produced by TH1 cells remained to be defined.  In an interesting follow-up study by the same group published in the February 21, 2013 issue of Nature, Braumüller et. al, further explore the mechanisms by which IFNγ and TNF produced by TH1 cells induce direct tumor cell growth arrest.

Culturing of pancreatic β-cancer cells with IFNγ plus TNF directly induced tumor cell growth arrest in the G1/G0 phase.  Interestingly, even following removal of IFNγ and TNF, tumor cells remained growth arrested for at least two weeks in vitro, indicating IFNγ and TNF induced cellular senescence.  Both IFNγ and TNF were required to induce senescence as either alone was not sufficient.

Induction of the p16INK4a gene by the combined actions of IFNγ-STAT1 and TNF-TNFR1 pathways was found to mediate this effect via consequential hypo-phosphorylation of the p16–retinoblastoma protein (Rb), thus maintaining its activated state.   Rb mediates senescence growth arrest by suppressing E2F, a transcription factor that promotes expression of cell cycle progression genes.  The role for these pathways was further validated by short hairpin (sh)-RNA knockdown of p16INK4a and p19 which inhibited tumor cell senescence by IFNγ and TNF.  The authors further demonstrated this phenomenon of TH1 cell – IFNγ and TNF induced tumor cell senescence in multiple cancer cell types as well as in an in vivo pancreatic cancer model.  Tumor cells rendered senescent by IFNγ and TNF in vivo remained arrested, even in the absence of T cells, B cells, and NK cells, following implantation into NOD–SCID/IL2rγ−/− mice.

Thus these studies define a mechanism of tumor-growth inhibition by the direct actions of the CD4+ TH1 cytokines IFNγ and TNF in mediating tumor cell senescence through activation of the Rb pathway.

The role of IFNγ in mediating tumor-immune responses is increasingly complex. IFNγ production has been negatively correlated with effective anti-tumor CD8+ T cell responses in some models (Gattinoni et. al) and also has been shown to induce expression of the immune inhibitory receptor PD-L1 (Lyford-Pike et. al).  Thus, these studies highlight that the contextual roles of immune cell effector cytokines are critical in their functions for regulation of tumor immunity and direct effects in tumor cells themselves.

Further Reading:

T-helper-1-cell cytokines drive cancer into senescence.  Braumüller H, Wieder T, Brenner E, Aßmann S, Hahn M, Alkhaled M, Schilbach K, Essmann F, Kneilling M, Griessinger C, Ranta F, Ullrich S, Mocikat R, Braungart K, Mehra T, Fehrenbacher B, Berdel J, Niessner H, Meier F, van den Broek M, Häring HU, Handgretinger R, Quintanilla-Martinez L, Fend F, Pesic M, Bauer J, Zender L, Schaller M, Schulze-Osthoff K, Röcken M. Nature. 2013 Feb 21;494(7437):361-5. doi: 10.1038/nature11824.

TNFR1 signaling and IFN-gamma signaling determine whether T cells induce tumor dormancy or promote multistage carcinogenesis.  Müller-Hermelink N, Braumüller H, Pichler B, Wieder T, Mailhammer R, Schaak K, Ghoreschi K, Yazdi A, Haubner R, Sander CA, Mocikat R, Schwaiger M, Förster I, Huss R, Weber WA, Kneilling M, Röcken M. Cancer Cell. 2008 Jun;13(6):507-18.

Cellular senescence: when bad things happen to good cells. Campisi, J. & d’Adda di Fagagna, F. Nature Rev. Mol. Cell Biol. 8, 729–740 (2007).

Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells.  Gattinoni L, Klebanoff CA, Palmer DC, Wrzesinski C, Kerstann K, Yu Z, Finkelstein SE, Theoret MR, Rosenberg SA, Restifo NP. J Clin Invest. 2005 Jun;115(6):1616-26.

Evidence for a role of the PD-1:PD-L1 pathway in immune resistance of HPV-associated head and neck squamous cell carcinoma.  Lyford-Pike S, Peng S, Young GD, Taube JM, Westra WH, Akpeng B, Bruno TC, Richmon JD, Wang H, Bishop JA, Chen L, Drake CG, Topalian SL, Pardoll DM, Pai SI. Cancer Res. 2013 Jan 3.

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