Cancer Immunotherapy involving Natural Killer Cells and Adenosine.

Tumor immunology is a very interesting subset of immunology as the dynamics of the immune system-tumor relationship is a long and very complicated one. Over years and years of cat-and-mouse, many immune evasion techniques have been discovered by researchers. One avenue of evasion is the release of immunosuppressive compounds by the tumor. Although tumor cells release molecules that can be recognized by the immune system and trigger a response (i.e. tumor-specific antigens (TSAs) and tumor associated antigens (TAAs)), these cells are not easy to detect. The tumor cells further ensure that these antigens are not recognized by release of immunosuppressive cytokines like IL-10 and TGF-beta (Mak and Saunders, 2011).

http://en.wikipedia.org/wiki/Metastasis

Another immunosuppressive compound is Adenosine, which is a useful molecule in the body as it is a DNA building block, a component of the main energy source in the body (ATP), and can be used to signal through one of its many receptors (A1, A2A, A2B, and A3). So it goes without saying that adenosine is present, in some form, all over the body.

Previous to the study by Beavis et al (2013), adenosine has been know to be produced from the breakdown of AMP by CD73 - a marker present on many types of cells. It has also been shown that anti-CD73 has resulted in delays in tumor expression, altogether rejection of tumor grafts in mice, inhibiting de novo carcinogensis, and preventing/reducing metastasis. In order to elucidate how exactly this immunosuppressive compound, adenosine, was suppressing tumor growth and metastasis, the authors set out to investigate adenosine in depth.

NK ALERT! New Subpopulation Linked in Restriction of B-Cell Transformation by EBV

EBV, or Epstein-Barr Virus, is a pretty common and current virus that affects approximately well over ninety percent of the human adult population. The virus belongs to the herpes family meaning after the bodies defenses respond to the initial viral lytic infection, the virus remains latent in B cells till further proliferation. The virus enters our tonsils after initial transmission via the saliva¹.

Usually, in the vast majority of infected individuals, virus induced CD8⁺ T cells are responsible for the main fight against EBV infection. However, Natural Killer (NK) cells play an important role for the initial innate immune response against EBV. This paper, A Distinct Subpopulation of Human NK Cells Restricts B Cell Transformation by EBV, further explores the abilities of a specific subpopulation of NK cells in preventing B cell transformation by EBV. The specific NK subpopulation, known as CD56brightNKG2A+CD94+CD54+CD62L2, was seen by researchers to produce an interestingly high amount of IFN-γ³. IFN-γ is an important cytokine that is essential for innate and adaptive immunity when dealing with viral and intracellular bacterial infections. The importance of IFN-γ in the immune system stems in part from its ability to inhibit viral replication directly, and most importantly for its immunostimulatory and immunomodulatory effects.³ So what does this mean?

T Cells take their shots, but Tumor Cells are too Much to Handle

Many types of cancers exist today in this world. One of the most common types of cancer found in humans is lymphoma. Lymphoma is a type of cancer where the white blood cells called the lymphocytes divide uncontrollably. (3) This form of uncontrollable division leads to what are called tumors. Tumors as most of us know are masses of abnormal cells due to an abnormal growth or division of cells.  The tumor cells are your Villains because they are trying to kill you. In the event of tumor cells attacking, the immune system recruits what are called T cells. The T cells, specifically Cytotoxic T Lymphocytes (CTLs), are the Heroes because they are trying to save you. In a new study, researchers examined the role of CTLs in a type of lymphoma called non-Hodgkin lymphoma, which is more common than the Hodgkin lymphoma. Although knowing the differences is not important in being able to understand the topic of this recent study, the differences between the two lymphomas can be found in the reference of lymphoma. (3) The importance of this paper lies within the interaction of the Cytotoxic T Lymphocytes and the lymphoma tumor cells. The new study recently published in The Journal of Immunology reveals how non-Hodgkin lymphoma tumor cells can actually “stop” the T cells. When we thought things couldn’t get any worse when it comes to cancer. When I mean by “stop”, the amount of tumor cells present determines a threshold at which the CTLs inactivate as well as delete rapidly in an antigen specific manner. Within the study, the researchers try to figure out the causes of how and what inactivates the CTLs.

 

Before we begin with the paper, here are some quick basics of immunology as well as what the research paper will be about to get a better understanding:

CTLs are called Cytotoxic T Lymphocytes, or CD8 T cells. They are called CD8 because they carry a surface marker protein called CD8 that allows them to recognize a surface protein called MHC class I (a protein expressed by all nucleated cells that presents broadly-specific foreign peptides). For understanding purposes of the T cells, I will be referring the T cells as CTLs just as the researchers call them in the paper. Like I said before, CTLs are a type of white blood cell found in the immune system. They kill cancerous cells, host cells that are infected by viruses, or cells that are damaged in any way. The purpose of having CTLs in your body is so that they monitor your body to destroy the person’s infected/irregular cells by direct cell contact before infected cells can spread throughout your body.

The CTLs kill the tumor cells in a process called apoptosis (same process for killing infected cells) where the tumor cells commit suicide after binding their antigens found on the surface to the TCRs (T Cell Receptors). Depending on the structure of the proteins in the binding sites found between the receptor and antigen, determines the specificity of the interaction. The interaction between an antigen and a receptor is like the binding of a lock and key.

Natural Killing and Adaptive Immunity: the role of NK cells in CD8+ T cell differentiation

In the human immune response, an encounter with a pathogen results in the rapid division of immune cells which neutralize and kill viruses, bacteria and parasites. While this rapid increase in the number of immune cells in the body is necessary for a robust and effective immune response, once the pathogen has been cleared it is essential that immune cell levels be brought back down to normal. CD8⁺ T cells, also known as cytotoxic T lymphocytes (CTLs), are one type of immune cell that follows this pattern of expansion and contraction. In the presence of an antigen, CD8⁺ T cells specific for that antigen divide rapidly, seeking out and killing infected cells and pathogens. When the threat has been cleared, high levels of CD8⁺ T cells specific for a single antigen are no longer necessary, and the majority of CD8⁺ T cells die by apoptosis. The surviving CD8⁺ T cells differentiate into memory T cells, allowing the body to respond more quickly and effectively the next time it encounters the same antigen. This process is essential for maintaining immunity to diseases; the memory T cells and B cells produced after an initial exposure are the reason that, for example, you only get chicken pox once. Similarly, memory T cells and B cells enable the body to prevent infection by pathogens against which it has been vaccinated.

In a paper published in the Journal of Immunology just this week, Soderquest, et al. examine the relationship between CD8+ s and natural killer cells (NK cells) after an infection has been cleared. NK cells are an important part of innate immunity – that is, the rapid and general immune response that includes both physical barriers to infection (i.e. the skin and mucosa) and non-specific killing cells like the NK cell. During the innate immune response, NK cells identify and kill cells that are either infected with viruses or are tumorigenic. Apart from their role in the innate immune response, recent studies have shown that NK cells play an important part in the development of the more specific adaptive immune response, particularly in promoting the differentiation of CD4⁺ “helper” T cells (Th cells). In investigating the relationship between CD8⁺ T cells and NK cells, Soderquest et al. focused on the NKG2D pathway of NK cell killing. NKG2D is a protein expressed on the surface of NK cells that binds to NKG2D ligand (NKG2DL) on a target cell and induces apoptosis via the perforin/granzyme pathway. The authors of this article hypothesized that NK cells influence the development of CD8⁺ T cell-mediated adaptive immunity by killing activated CD8⁺ T cells via the NKG2D/perforin pathway.

Interfering with Pancreatic Cancer Growth – Role of Interferon Regulatory Factor-2 in Pancreatic Tumorigenesis

Apple CEO Steve Jobs; 2011 Nobel Prize Laureate Ralph Steinman; actor Patrick Swayze; actor Michael Landon; opera singer Luciano Pavarotti; comedian Jack Benny; actress Donna Reed; musical composer Henry Mancini. These well-known individuals, despite varying career paths, share a rather tragic connection: each has lost his or her life as a result of pancreatic cancer. Pancreatic cancer, or pancreatic carcinoma, has the highest mortality rate amongst all major cancers. Within 5 years of diagnosis, approximately 94% of patients will die from this form of cancer. Just this year, it is estimated that 44,030 cases of pancreatic cancer will be newly diagnosed, while approximately 37,660 patients will have died as a result of the disease [2]. Cigarette smoking, obesity, type II diabetes, blood types A/B/AB, and loosely defined genetic influences may present elevated risk for developing pancreatic carcinomas; however, the majority of patients diagnosed with pancreatic cancer do not exhibit any identifiable risk factors. Pancreatic cancer is a rather aggressive malignancy, and the high mortality rate is attributed to the difficulty in detecting the cancer at an early stage, as well as to the complexity involved in its development. Because this form of cancer has frequently reached advanced stages by the time of diagnosis, removing the tumor through surgery is not often a treatment option. When the tumor has spread beyond the pancreas (metastasized) and is not operable, chemotherapy alone, or in combination with radiation therapy, is utilized. Traditionally, the chemotherapy drug selected for treatment is gemcitabine [3]. Such limited and largely unsuccessful therapeutic options available for pancreatic cancer patients underscores the importance of defining new mechanisms for treatment.

Combination Therapy--Giving Cancer the 1-2 Punch

It has been well documented that many cancer cells over express Intreleukin-13 receptors—in particular 4T1 breast carcinoma cell lines and MCA304 sarcoma cell lines (Joshi, 2000). Interleukin-13 (IL-13) is a small, cell-signaling protein that is derived from a Th2 immune response that binds to Interleukin-13 receptors (IL-13R). IL-13Ralpha2 is one of two receptor subunits of the IL-13R complex. This subunit binds IL-13 with high affinity to mediate a signal transduction, ultimately resulting in the production of TGF-beta (Fichtner-Feigl et al., 2006).

Recent studies have focused on targeting IL-13R of tumor cells in order to control the progression of cancer. For this reason, an immunotoxin, IL13-PE, was formulated by recombining IL-13 and Pseudomonas exotoxin in order to target IL-13R expressing tumor cells (Kawakami et al., 2001). IL13-PE is able to specifically recognize IL-13Rs, limiting toxicity to cancerous tissues (Puri et al., 1996). IL13-PE acts to inhibit protein synthesis and cause cell death of IL-13 expressing cells by irreversibly ADP-ribosylating the diphthamide residue of elongation factor 2 (Fichtner-Feigl et al., 2006).

Vaccines have also been utilized as an alternative method to induce a strong immune response against tumor cells. T cells and B cells are leukocytes that are responsible for regulating the immune response by recognizing harmful entities in the body and carrying out their destruction. Antitumor vaccines are intended to incur an antigen-specific immune response against a tumor peptide by priming the immune system to activate T cells and B cells (Nakashima et al., 2010). By stimulating the production of T cells and B cells to recognize tumor cells, the proliferation of tumors can be controlled. One particular vaccine, IL-13Ralpha2 DNA vaccine, has shown to be effective in limiting tumor growth in both 4T1 breast carcinoma cell lines and MCA304 sarcoma cell lines (Nakashima et al., 2010).

STAT3β: Not Such A Downer

STAT3 is a transcription factor that has been of particular interest to oncologists lately for its promotion of oncogenesis through constitutive activation. STAT3 has two key residues that must both by phosphorylated by a kinase in order for the protein to dimerize into a homodimer or heterodimer and thus be active. The two residues, tyrosine 705 and serine 727, are both found on exon 23. STAT3 is activated by several interferons, cytokines, and growth factors.

An article published in the October 25, 2011 edition of PNAS explored the study of Dr. Francesa Zammarchi et al on the alternative splicing that leads to two isoforms of STAT3: STAT3α and STAT3β. Exon 23, which includes the two amino acid residues necessary for activation of the STAT3 TF, can be fully or partially included in the final product. If the entire exon is included, STAT3α is formed and the protein is fully functional. If the 55 amino acids at the end of exon 23 are excluded from the final product, then STAT3β is made. The premature termination of the protein causes serine 727, a necessary residue for phosphorylation and activation of the STAT3 protein, to be truncated. This renders STAT3β able to maintain its DNA binding function, but not able to be fully active as a transcription factor.

Previously, it has been thought that STAT3β was a dominant-negative regulator based on its ability to bind DNA and thus competitively inhibit the activity of STAT3α. However, the findings of Zammarchi et al are questioning that designation, and have made a strong case for STAT3β as an antitumorigenic factor with its own specific set of regulated genes and proteins.

In a series of experiments that tested STAT3β regulation of both RNA and protein products, Zammarchi et al found that IL-8, LEGDF, and PCAF were all regulated specifically by STAT3β rather than total STAT3 knockdown. The transcription factor was demonstrated to downregulate both LEDGF and PCAF transcription and translation. IL-8 is involved in inflammation and in the chemotaxis of neutrophils, which are innate immune cells that phagocytose pathogens. LEDGF is a chromatin-binding protein and a transcriptional coactivator, as well as a pro-survival growth factor. PCAF is a histone acetyl transferase and transcriptional coactivator that promotes growth, invasion, and drug resistance. Clearly these genes are all of great interest to oncologists as potential cancer treatment targets, and thus STAT3β is coming into focus as a gene and protein of interest for all those involved in fighting cancer.

It has been previously demonstrated by Yue and Turkson1 that overexpression of STAT3β can induce apoptosis and inhibit tumor growth, and now the next step investigating the reasons for these effects has begun. Zammarchi et al showed that the STAT3β regulation occurs in several different types of cancer cell lines, including breast cancer, prostate cancer, and lung cancer, demonstrating the potential widespread nature of this factor as a cancer treatment target. There is still a long list of topics to be further investigated before STAT3β can be implicated as a prospective treatment target in humans including determining side effects, testing in animal models, and the development of drug delivery systems that are compatible with this substance, but nonetheless STAT3β appears to be a promising target for future oncological studies.

Reference:

Zammarchi, F. et al. (2011) Antitumorigenic potential of STAT3 alternative splicing modulation. PNAS 108(43):17779-84.

Additional Citation:

1) Yue, P., Turkson, J. (2009) Targeting STAT3 in cancer: How successful are we? Expert Opin Investig Drugs 18:45-56.

Treating γδT cells as Individuals May Help Develop New Cancer Treatments

γδ T cells are a subset of T cells that bridge innate and adaptive immunity. γδ T cells bind and recognize a broad range of foreign materials with their “γδ” receptors. These foreign entities can include proteins and lipids from pathogens as well as host stress molecules. This is the innate part of their function; it is fairly non-specific. They then generate either CTL-like (toxic) or Th-like (helper) responses against these pesky materials. This is the adaptive part of their function; it is more specific (1). Often, it is thought that all γδ T cells behave similarly, in the way that was just described.

A growing body of evidence, however, including a recent paper by Yin et. al. (2), suggests that different subsets of γδ T cells perform unique functions (3). In their work, Yin and coworkers found that one type of γδ T cell, Vg1 γδ T cells, inhibits the antitumor response of another type of γδ T cell, Vγ4 γδ T cells. (Vγ1 and Vγ4 refer to a particular gene segment that codes for part of the receptor chain; think of it as a receptor flavor).

In a previous study (4), Yin and coworkers found that Vγ4 γδ T cells killed tumors using INFγ (a signaling molecule that can: tell other cells to kill the tumor, prevent blood and nutrient flow to the tumor, and prevent spreading of the tumor by constructing a barrier around it (1)) and perforin (a molecule that kills a tumor by poking holes in it (1)). In this study, Yin et al. found that Vγ1 γδ T cells inhibit the anti-tumor response of Vγ4 γδ T by using signaling molecule IL-4.

Yin and co-workers placed Vγ1 γδ T cells , Vγ4 γδ T cells, and tumor cells in mice in different combinations and monitored tumor growth. Vγ4 γδ T cells alone suppressed the tumor. Addition of Vγ1 γδ T cells reduced this suppression. They obtained similar results when they performed the experiments in vitro (outside of mice, in culture).

When they separated the two types of cells by a membrane in vitro, they observed the same loss of tumor suppressor function. This suggests that the Vγ1 γδ T cells do not depend on direct cell-to-cell contact to inhibit Vγ4 γδ T cells and instead depend on a soluble mediator. This conclusion was further confirmed by a dye staining experiment that showed that the two types of cells localized to different parts of the tumor and were therefore unlikely to directly contact each other at all.

The soluble mediator molecule was determined to be IL-4. Suppressing IL-4 restored Vγ4 γδ T tumor suppression abilities. Furthermore, adding IL-4 alone, but not Vγ1 γδ T cells, to Vγ4 γδ T cells suppressed antitumor properties. The relationship between the two cell types came full circle when Yin and coworkers found that IL-4 down regulates NKG2D (Natural Killer Group 2 receptor, common to NK cells and γδ T cells), a receptor that tells Vγ4 γδ T cells to make IFNγ and perforin. IL-4 interferes with IFNγ and perforin. Thus, molecules made by Vγ1 γδ T cells interfere with molecules made by Vγ4 γδ T cells.

The study leaves some questions unanswered. For example, why do the different γδ T cell subsets localize to different parts of the tumor? How does IL-4 down regulate NKG2D receptors? More broadly, how do Vγ4 γδ T and Vγ1 γδ T cells interact with other cell types? But it also provides novel and potentially useful results. It suggests that Vγ1 γδ T is a regulatory T cell of sorts in that it dampens the response of another type of T cell, Vγ4 γδ T. The study also opens the door for a possible tumor therapy where Vγ1 γδ T cells are selectively killed or inactivated so that Vγ4 γδ T cells can be allowed to carryout out their tumor-killing mission.

Reference:

Yin, Z. et. al. 2011. Regulatory Role of Vγ1 γδ T cells in Tumor Immunity through IL-4 Production. J. Immunol. 187: 4979-4986.

Additional Citations:

(1) Mak, Tak and Mary Saunders. Primer to the Immune Response. California: Elsevier, 2011. Print.

(2) Yin, Z. et. al. 2011. Regulatory Role of Vγ1 γδ T cells in Tumor Immunity through IL-4 Production. J. Immunol. 187: 4979-4986.

(3) Born,W. K., Z. Yin, Y. S. Hahn, D. Sun, and R. L. O’Brien. 2010. Analysis of γδ T cell Functions in the Mouse. J. Immunol. 184: 4055–4061.

(4) He, W. , J. Hao, S. Dong, Y. Gao, J. Tao, H. Chi, R. Flavell, R. L. O’Brien, W. K. Born, J. Craft, et al. 2010. Naturally Activated Vγ4 γδ T cells Play a Protective Role in Tumor Immunity through Expression of Eomesodermin. J. Immunol. 185: 126–133.

Men: Beware the ETS Family of Transcription Factors-It May Control Immunity Genes Involved in Prostate Cancer

Cancer is a state of abnormal and uncontrolled cell growth that can potentially spread throughout the body. Cancerous cells have adverse effects, which the body has many defenses against in order to prevent tumor growth and cancer development. The immune system is thought to aid in the suppression or promotion of tumor growth by exerting multiple cellular controls over the death or proliferation of certain cells (Lin and Karin, 2007). With vast numbers of people being affected by cancer on a daily basis, it is important to identify cellular differences between cancerous and healthy states. Identifying changes that might be associated with the onset of cancer proliferation can potentially lead to better understanding of the onsets of cancer.

The prostate, a gland found in males that stores liquid that carries sperm, is highly susceptible to inflammation and cancer (Eisenburg Center, 2005). Prostate cancer is becoming increasingly common in adult males: In fact, in 1990 prostate cancer became the most frequently diagnosed cancer in men, constituting 29% of all cancers found in men (American Cancer Society, 2011). With such alarming numbers of incidence, prostate cancer has become a great health concern of epidemic proportions. However, the theory behind the recent drastic increases may not be solely attributable to an increase in the prevalence of the disease, but instead to new tests, such as the PSA (prostate-specific antigen) test. The PSA test allows for more accurate and determinate prostate cancer detection (Levy, 1995). Even though the higher number of reported cases appears daunting, the PSA test has had the positive effect of detecting the cancer earlier for effective treatment, resulting in decreasing mortality rates. Therefore, PSA tests may allow for prostate cancer to be detected at younger ages, resulting in more healthy, robust men being diagnosed with greater chances of survival with treatment (Hsing, Tsao, and Devesa, 2000).

Dendritic Cells Directly Engage Cancer

As members of the Colgate community know, especially in light of the recent passing of student Vic Krivitski ’12, cancer affects everyone’s life. Less apparent are the specific ways in which the immune system acts to mitigate cancer. To elucidate these mechanisms, a recent study in the Journal of Immunology investigates the role dendritic cells (DCs) play in killing cancer cells.

Cancer is a condition of uncontrolled cell growth. Not only are cancer cells good at promoting their own proliferation, but also they are proficient at avoiding the immune system. DCs are immune cells that have two characteristic functions, both of which can be thwarted in the presence of cancer (1). Typically, DCs act as antigen presenting cells (APCs), presenting antigens, pieces of pathogens, to lymphocytes to activate them. They can also secrete cytokines, signal molecules, that tell other cells to combat pathogens. Less well understood is DCs’ ability to act as killer DCs (KDCs), by directly engaging pathogens, and how this function is affected by cancer.

Larmonier and Bonnotte et. al. attempt to enhance the understanding of human-derived KDCs (hKDCs). KDCs have been examined previously in rats, mice, and humans (2,3,4) but this investigation provides finer details of hKDCs’ effective activation, mechanism for inducing cancer cell death, and ability to retain characteristic DC function.

hKDCs were prepared by exposing monocyte-derived DCs from cancer-free and stage IV cancer participants to low concentrations of an activating molecule, LPS. These hKDCs were found capable of killing several tumor lines (including HeLa) and reducing cancer cell viability up to 80%. This is a particularly exciting result because DCs derived from cancerous environments were found to be as effective as those from cancer free environments.

The mechanism for cancer cell death was elucidated via several experiments. ESR spectroscopy, which measures free radicals, showed that hKDCs produce reactive oxygen species (ROS). Decreased anti-tumor activity in the presence of FeTPPS, a free radical inhibitor, confirmed this result. When cancer cells and hKDCs were separated by a semi-permeable membrane, decreased anti-tumor behavior was observed suggesting that hKDCs kill cancer cells via direct cell-cell contact. Additionally, a staining experiment that allowed visualization of the dying cells as well as the death of certain apoptosis-resistant cancer cells suggests that the cells die by necrosis (catastrophic cell death) rather than apoptosis (programmed cell death). This, too, is promising because cancer cells are particularly adept at avoiding apoptosis, so a treatment that includes a necrotic mechanism might be effective against these cells.

Furthermore, the authors demonstrate that hKDCs retain their original DC functions: they are still able to present antigens (even those from cancer cells they kill themselves!) and produce cytokines. These promising results are not problem free and will require further study. hKDCs’ ability to kill certain non-cancerous cells requires an extensive investigation of the interactions between hKDCs and healthy cells. (It is important to note, however, that T lymphocytes were not killed by hKDCs. In fact, hKDCs produce cytokines that help T lymphocytes resist death.) Similarly, the mechanism of cell death itself is a danger to healthy cells, because necrosis releases dangerous toxins. Nevertheless, this study makes strides in understanding the relevancy of hDKCs for potential cancer treatments and may aid in the development of such treatments.

Reference: Lakomy, D., Janikashvili, N., Fraszczak, J., Trad, M., Audia, S., Samson, M., Ciudad, M., Vinit, J., Vergely, C., Caillot, D., et al. 2011. Cytotoxic dendritic cells generated from cancer patients. J. Immunol. 187(5): 2775-82.

Other Citations:

(1) Brown, M.P., Diener, K.R., Fraser, C.K., and Hayball, J.D. 2010. Unraveling the complexity of cancer immune system interplay. Expert Rev. Anticancer Ther. 10: 917.

(2) Fraszczak, J., Trad, M., Janikashvili, N., Cathelin, D., Lakomy, D., Granci, V., Morizot, A., Audia, S., Micheau, O., Lagrost, L., et al. 2010. Peroxynitrite-dependent killing of cancer cells and presentation of released tumor antigens by activated dendritic cells. J. Immunol. 184: 1876–1884.

(3) Chauvin, C., Philippeau, J. M., He ́mont, C., Hubert, F. X., Wittrant, Y., Lamoureux, F., Trinite, B., Heymann, D., Re ́dini, F., and Josien, R. 2008. Killer dendritic cells link innate and adaptive immunity against established osteosarcoma in rats. Cancer Res. 68: 9433–9440.

(4) Hill, K. S., Errington, F., Steele, L. P., Merrick, A., Morgan, R., Selby, P. J., Georgopoulos, N. T., O’Donnell, D. M., and Melcher, A.A. 2008. OK432- activated human dendritic cells kill tumor cells via CD40/CD40 ligand interactions. J. Immunol. 181: 3108–3115.