Friday, October 4, 2013

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.

Here is some background info on what is involved in the paper. Reading this would be extremely helpful in understanding what is going on:

The scientists start with two transgenic mice to model the human non-Hodgkin lymphoma tumor cell groups with the first being Eμ-myc-GFP, and the second being Eμ-myc-OVA.

Allow me to clarify the meaning behind the names for the two groups. Firstly, the “Myc” is a gene being used to research the study. The purpose of the Myc in our body is so that the Myc gene codes for a protein that acts as a transcription factor that binds to regions of DNA to regulate expression of other genes. When Myc is mutated, it causes overexpression of other genes such that it ultimately leads to cancer. How these scientists are able to manipulate the gene in order to mutate the Myc is by having an enhancer called the IgH enhancer. Myc is a proto oncogene. (4) The IgH enhancer, represented as the “Eμ”, acts on the Myc to develop into lymphoma cancer cells so that the researchers can study the causes of lymphoma tumor cells and CTL inactivation inside transgenic mice.

“OVA” is the abbreviated form of ovalbumin. Ovalbumin is the main protein found in egg white and it is commonly used as an antigen in immunology to stimulate an immune response to test whether a specific immune cell will elicit a response compared to a control.

The control simply contains GFP along with the tumor cells. GFP For those who do not know what GFP stands for is Green Fluorescent Protein. GFP is used frequently in biology labs in order to see if that protein will actually give a color as an indicator of its presence.

The Eμ-myc-GFP contains tumor cells with GFP, but no OVA mouse strain. This is the control. The Eμ-myc-OVA also contains tumor cells, but with the addition of both GFP and OVA to see if the OVA is a functional antigen.

The two groups both contain tumors; however, only the OVA cell group will elicit an immune response because only that cell group contains an antigen that will bind to the CTLs that are specific for OVA.

Now that you have some context, knowing the function of the “Eu-Myc” in both groups is not as important as knowing the “GFP” and “OVA” parts because the OVA determines the antigen binding to the CTL in this paper. So pay attention to the CTL interaction when you are reading.

Now we are getting back to the subject of matter. The two groups both contain tumors; however, only the OVA cell group will elicit an immune response because only that cell group contains an antigen that will bind to the CTLs that are specific for OVA antigen. In this paper, the CTLS are looking to kill the tumor cells that contain ovalbumin. Those CTLs are specified as anti-OVA OT-I CTLs because they are specific for the ovalbumin.

When the scientists injected the anti-OVA CTLs after 3 days of tumor development, the CTLS killed off almost all of the tumor cells. But when the scientists injected the anti-OVA CTLs after 5 days of tumor development, the tumor cells did not die off. In fact, the OVA tumor cells in the 5-day count were about the same high numbers as the control group in days 3 and 5.  In the 3 days and 5 days of the GFP control, the tumors stayed relatively the same with high numbers of small increases after each day due to cell division as expected because there was no CTL specific for the control. (Fig 1)

After experimenting the interactions between the CTLs and the tumor cells, the scientists recovered the amounts of the CTLs in each group for both days from the spleen where all the action took place. In order to see any type of difference in the CTL counts for the 3 and 5-day counts for each group, lymphocytes were identified and measured via a process called flow cytometry. Upon recovering the CTLs, only a few CTLs were recovered from the Day 5 of the GFP-OVA mouse, but the CTLs in the GFP alone mouse were still the same high population number compared with the day 3 results, and compared to the no-CTL injection results. (Fig 2)

The results were promising, but the scientists needed to dive in deeper into how the CTLs were being deleted and inactivated.

            From recovering the CTLs, the scientists also noticed the characteristics of exhaustive T Cells and increased PD-1 receptors. Exhausted CTLs are as stated “a process that involves the progressive loss of function and ultimately death of the virus-specific T cells”. (2) The other characteristic that scientists noticed were that the day 5 CTLs showed higher numbers of having PD-1 (Programmed Cell Death 1) receptor and down regulation of TCR expression. The scientists realized then that the cause of the up and down regulation of the receptors had to be related to antigen specific.

They knew the CTLs were antigen specific because of the OVA. In order to confirm if this antigen specific was caused by direct cell contact or indirect contact by cytokines, they also injected a CTL that had characteristics of being a nonspecific antigen such as glycoprotein. The CTL is named gBT-I CTL, which also expresses receptors that could be affected by the inactivation as well as the OVA CTLs only if the mechanism is by the release of cytokines. After the scientists co-injected the two CTLs, they found that only the OVA CTL was affected. This meant the inactivation of CTLs is caused by direct contact of the antigen because the gBTI-CTLs were unaffected. The scientists stated that the direct contact also meant the antigen is a cognate antigen that requires an APC (antigen presenting cell that presents the specific antigen to the CTL).   

Now that the researchers have figured out precisely how the inactivity of CTLs occurs as that being direct antigen specific, the question becomes what causes the inactivity. Many of the researchers thought it could be the likes of an APC.

            After figuring out the dendritic cells were not the cause of the inactivation when they put dendritic cells (a cell type of an APC) that were deficient in presentation with CTLs into tumor bearing mice, they next assessed whether the tumor cells were the APCs that inactivated the CTLs. The scientists injected a new lymphoma called bm1.Eμ-Myc-OVA that is deficient in OVA presentation meaning that the CTL should live if the tumor is unable to make direct contact with the CTL. When the scientists gathered the results for the tumor cells, the CTLs were a smaller number in the original OVA while the CTLs in the bm1 deficient presentation of OVA were at the same amount as those CTLs in the GFP control meaning the CTLs lived. This showed that the CTLS were active, but the tumor cells still had the same population, as those in the GFP control because the CTLs were unable to make contact to try to kill the tumors since the tumors were deficient in peptide presentation. 

            Afterwards, the scientists then tried to see if the tumor cells resisted the chemicals the CTLs release when they engage the tumor cell. The chemicals the CTLs release are called interferon- γ, and kill the tumor cells. To see if the tumor cells acquired a resistance after the contact inactivation of CTLs, they injected more CTLs a day after the 5 -day count at a smaller tumor population. As a result, the tumors ended up dying meaning that the tumors did not acquire resistance to CTLs after the initial contact for inactivation of CTLs. Since tumor cells do not create a resistance, then the tumor cells can inactivate the CTLs at a certain threshold.

Now the scientists test to see what affect the chemotherapy drug CTX, also called cyclophosphamide, has on the tumor cells with CTLs. The scientists wanted to compare the affects of a low dose and high dose of the chemotherapy on the tumor cells to see if the CTLs inactivate depending on the dosage level. When the scientists injected CTLs after a low dose, the tumor cells quickly bounced back to eventually inactivate the CTLs. But, when the scientists placed CTLs after a high dosage of chemotherapy on a high population of tumor cells, the CTLs were effective in eliminating the rest of the lymphoma tumor cells. This showed the CTLs become inactive at a certain threshold, but at the same time show another treatment that is successful in eliminating tumor cells. Now that they identified the problem for the most part………what is next?

Even though these scientists did not find the exact answer they were looking for in terms of the exact mechanism for inactivation, they did find a more efficient treatment for patients with lymphoma, and that is by supplying CTLs directly after the chemotherapy so that the large tumor counts are reduced enough to a point where they will no longer be able to reach the threshold to inactivate the CTLs.

The researchers noted that uninfected host cells carry mechanisms to inactivate the activity of CTLs so that the CTLs will not damage any of the individual’s vital tissues (as a tolerance mechanism). Given that, the researchers believe the CTLs are being inactivated due to an autoimmune prevention mechanism. As a result of this mechanism, it appears that the mechanism is still preserved within the tumor cells since the tumor cells used to be normal host cells before they became tumor cells. As a result of that hypothesis, it would be interesting to further the research by comparing those CTLs that recognizes viruses/tumors being compared to those anti-self CTLs to see if the anti-self CTLs will inactivate as an autoimmune prevention when it is in the presence of tumor cells.   

 Primary Source:

1)Prato, Sandro et. Al. Rapid Deletion and Inactivation of CTLs upon Recognition
of a Number of Target Cells over a Critical Threshold. 9 September 2013. Journal Immunology, 191, 3534-3544. Online Publication.

Secondary Sources:
T Cell exhaustion:




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