Revolutionizing Immunology One T Cell at a Time

T cells…the cure to cancer?

The immune system is attacked every day. Our cells are constantly defending our bodies from bacteria, viruses, and microorganisms.

How is it possible that every time we are attacked, we don’t get sick?

As we all know, there are front-line workers of clinics, hospitals, grocery stores, etc. who we must thank for their service everyday.

But what about our blood cells? Who are they? How do they become our body’s greatest warriors?

What is hematopoiesis?

Hematopoiesis (spelled with Greek root hema or haema) is the process of creating blood cells from stem cells. The word “trilineage” is often correlated with this process because red blood cells, white blood cells, and platelets are its outcomes.

When a zygote (a single-cell fusion of sperm and egg) becomes multicellular and its embryonic stem cell count reaches around 150, the organism is considered a “blastocyst.”

As the embryo develops, existing cells divide and produce new daughter cells because they undergo a process of distributing replicated chromosomes and cytoplasm domains called mitosis or more commonly, cell division.

The cells that make up the blastocyst are pluripotent, meaning they can transform into any type of cell in the body (muscle, skin, blood, nerve, etc.) or divide into more stem cells to continue the growth of the embryo.

The stem cells that can transform into blood cells are called multipotential hematopoietic stem cells (MHSCs). The haematopoietic differentiation tree is a diagram showing all the known, possible pathways of MHSCs.

By observing the diagram, you can see that the stem cell is posed with a fork in the road. The path in which the stem cell takes is completely based on the random expression of genes.

Where does it take place?

The yolk sac is a membranous sac just outside of an early-developing embryo. It is the location of hematopoiesis and is responsible for the embryo’s blood circulation.

Once the embryo develops into a fetus, the spleen, liver, and bone marrow become the new sites of blood cell production.

Once the child is born and matures into an adult, the production of blood cells decreases and the bone marrow becomes the main location of hematopoiesis.

What occurs in hematopoiesis?

As mentioned earlier, the MHSCs are the forerunners of hematopoiesis. These stem cells will become either a myeloid or lymphoid progenitor or an “ancestor” to the next stages of its maturity. MHSCs secrete a type of cytokine (a broad class of proteins involved in cell communication) called interleukins that determines whether it will turn into a myeloid or lymphoid cell.

The stem cell will secrete interleukin-3 to transform neighboring stem cells into myeloid cells or interleukin-7 to produce lymphoid cells.

Myeloid cells make up the baseline of a healthy human body’s amount of blood cells (red, platelets, and even some white blood cells).

Lymphoid cells are better known as white blood cells (or leukocytes). These white blood cells differentiate into T cells and B cells. They both are produced in the bone marrow.

T cells mature in the organ located above the heart: the thymus (T is for thymus). The thymus is a place where only T cells develop, making it the T cells’ primary lymphoid organ.

Thymus activity decreases as a person gets older which is why children tend to have physically bigger thymuses than adults or teenagers.

Differentiation continues in the thymus. There are different types of T cells with various responsibilities that result from its precise development stage.

How do T cells develop and differentiate?

T cell differentiation can be difficult to follow, so in order to fully comprehend the process, we will follow the maturation span of a T cell named Timmy.

Everyone say Hi Timmy!

Timmy has been produced in the bone marrow via hematopoiesis. He’s a few days old, but is determined to find a job in the real world. Timmy is released from the bone marrow where he grew up for a short amount of time and taken into the bloodstream. A capillary transports Timmy to the thymus.

Wait, wait, wait! How did Timmy know which organ to go to? After all, he’s only a baby!

The thymic cells of the thymus attracted Timmy with chemokines. Chemokines are another type of cytokines that can attract precursor T cells (like Timmy) to the sub capsular region of the thymus. Timmy enters this part of the thymus via high endothelial venules (HEVs) that act as lymphocyte pumps for certain organs. Timmy can be attracted by thymosin, thymotaxin, or even thymopoietin. This method of attraction is called chemotaxis.

But in order for Timmy to eventually obtain a job, he must undergo training or a series of events that alter Timmy’s genes. This entire training course will take three weeks.

*Note that the thymus is structured in layers, each layer specific to certain stages of Timmy’s training:

The chemokines that attracted Timmy, bound with his cell membrane’s receptor protein. This triggered a type of cellular response called gene expression.

You can learn more about the cell membrane and receptor proteins here.

Gene expression can occur when a ligand binds to its specific receptor protein on a cell membrane. This binding kickstarts a series of reactions, leading up to where proteins called transcription factors trigger a gene to “turn on” and eventually inducing protein synthesis.

Phase 1 — Production.

A specific set of Timmy’s genes were stimulated. This led to the synthesis of two enzymes called RAG1 (recombination activating gene 1) and RAG2 (recombination activating gene 2), both of which are a type of recombinase.

Recombinases “shuffle” DNA through a process called variable diversity joining or V(D)J recombination.

V(D)J recombination is very similar to a CRISPR tool because RAG1 and RAG2 cut and paste gene segments in order to produce T cell receptors (TCRs).

This can negatively impact Timmy because an error in recombination can result in malignant tumors, or positively impact Timmy by granting him genetic diversification in order to code for different TCRs for different antigens or foreign substances that trigger an immune response. Every antigen has a specific shape or key. Each TCR is like a corresponding lock to each antigen or key.

Timmy feels another tingling within his cytoplasm. The chemokines have triggered yet ANOTHER set of genes to be expressed. This cellular response led to the production of proteins called cluster differentiation proteins. Timmy produced the proteins CD4 and CD8, but he has the potential to produce different cluster differentiation proteins as well.

Timmy has completed what is called the “Double Negative Stage” aka Phase 1. The term “double negative” comes from the fact that Timmy initially started off with no rearranged DNA to make different TCRs such as TCRɑβ and TCRγδ.

*Note that other precursor T cells just like Timmy can produce other proteins such as CD25, CD44, or cKit, making them unique.

Timmy has now advanced in his training and is entering the “Double Positive Stage” (because he now has functional proteins and TCRs) in the cortex of the thymus. Timmy must undergo a series of potentially successful dates with some thymic cells.

Yes, I said dates.

But here’s the catch: these dates are no ordinary dates. If Timmy’s date goes wrong, he will undergo apoptosis, also known as cell suicide. The pressure is on.

Phase 2 — Positive Selection.

He meets up with thymic cell Tiana for his first date. He attempts to impress Tiana with his new cluster differentiation proteins, CD4 and CD8. If Tiana recognizes these proteins and deems them “hot and sexy” she will allow Timmy to live.

*Note that Tiana only needs to be slightly impressed. In biological terms, Tiana and Timmy must have only a low-affinity interaction or an interaction with low attraction. If Tiana is overly impressed or they have a high-affinity interaction, she will consider Timmy being capable of causing autoimmune diseases and will not allow him to move on to the next stage.

Hold on, how does Tiana determine the hotness and sexiness of Timmy’s CD8’s and CD4's?

Tiana had special molecules called major histocompatibility complexes, particularly MHC1 and MCH2. These molecules were able to recognize Timmy’s CD4 and CD8. This recognition is a good thing because a typical T cell should be able to recognize foreign antigens presented on the surface of any cell. The recognition of these surface proteins is called positive selection.

Tiana spared Timmy’s life. Timmy’s next test will determine his ability to recognize self-antigens or antigens that do not come from foreign organisms.

Phase 3 — Negative Selection.

Tiana begins the test by presenting a self peptide chain or self-antigens on the surface of her MHC molecules. As Timmy’s cluster differentiation proteins are already interacting with her MHC molecules, Timmy is being tested if his TCRs (T cell receptors) will recognize and bind to Tiana’s self-antigens.

If the TCRs do recognize the self-antigens as foreign and thus binding to them, Timmy must execute himself (apoptosis). A T cell must NEVER be able to consider self antigens as foreign or autoimmune diseases may emerge.

If his TCRs behave cooperatively, it means that his TCRs were a different shape than what could fit Tiana’s self-antigens and Timmy has completed the test. He has completed the stage called negative selection.

*Note that Timmy’s CD4 and CD8 proteins did not disappear, they are just not pictured. They are still interacting with Tiana’s MHC molecules!

Timmy’s last training session before he graduates Thymus Academy takes place in the medulla of the thymus.

Phase 4 — Graduation.

He must now randomly choose whether he wants to have one more date with Tiana or go on a date with another thymic cell. Timmy’s cluster proteins, CD4 and CD8, must pair with either an MHC1 or MHC2 molecule of another cell.

Tiana has more MHC2 molecules but another thymic cell has more MHC1 molecules. If Timmy’s CD4, with the correct orientation, decides to bind with Tiana’s MHC2 molecule and his CD8 does NOT interact with another cell’s MHC1 molecule, Timmy’s genes will “downregulate” or decrease the number of CD8 proteins on his cell membrane and increase the number of CH4 proteins (because in this case, Tiana is his type). This allows Timmy to conclude his training and graduate Thymus Academy and become a T helper cell!

Let’s say Timmy’s CD8 proteins manage to interact with another thymic cell’s MHC1 molecules. The same down-regulation and up-regulation process occurs, but instead, the number of CD4s will decrease and CD8s increase. Timmy’s cell membrane should now house ONLY CD8 proteins and no CD4s. This allows Timmy to become a T cytotoxic cell!

*Note that if Timmy is not able to have a low-affinity interaction with either MHC1 or MHC2 molecules or is able to have low-affinity interactions with BOTH, he will die via apoptosis. He must choose only one MHC molecule.

Even though Timmy has graduated from Thymus Academy, he can differentiate into a T regulatory cell as well. Certain cytokines such as interleukin-2 can induce T helper or T cytotoxic cells to differentiate into these T regulatory cells in order to prevent autoimmune diseases.

Thymus-Academy-alumni Timmy can choose to stay local or go out of state. If he wants to go out of state, he has the ability to inhabit the sinusoidal capillaries of the spleen or the cortex of a lymph node. If Timmy graduates as a T regulatory cell, he can choose to live out of state, or locally in Hassal’s (thymic) corpuscles, an abundant area of T regulatory cells in the thymus.

Let’s recap Timmy’s training:

1. Production: Chemokines induce Timmy to make recombinases or DNA-shuffling enzymes RAG1 and RAG2, cluster differentiation proteins CD4 and CD8, and different TCRs corresponding to specific antigens. This can also be referred to as the Double Negative Stage.

2. Positive selection: Timmy’s CD4 and CD8 proteins recognize Tiana’s MHC molecules. This can be referred to as the first part of the Double Positive Stage. Fun fact: only 10% of T cell precursors get through this stage.

3. Negative selection: Timmy’s TCRs do NOT recognize Tiana’s MHC molecule’s self-antigens. This can be referred to as the second part of the Double Positive Stage.

4. Graduation: Timmy can choose (randomly) to become either a cytotoxic T cell or helper T cell and live out of state. He even has the choice to become the former and then develop into a T regulatory cell and possibly live in the thymic corpuscles.

What are the types of T cells?

1. Helper T cells secrete cytokines or communication proteins after recognizing specific antigens on neighboring cells that activate other T and B cells. To trigger an immune response, helper T cells are extremely important when the body needs back up. These cells have many CD4 proteins on their membranes.

2. Memory T cells remain after a viral or bacterial attack. When the body’s immune system successfully kills off all infected cells, the remaining killer T cells or effector T cells become deactivated and die off in order to regulate the body’s immune balance.

Immune balance refers to the proper maintenance of how the body prepares and executes for pathogen-related fights and recoveries. If the majority of the effector T cells do not undergo apoptosis, they may cause damage to neighboring tissue. It’s like eating candy: having a little handful of candy is good for the soul, but having too much will lead to cavities and high blood sugar levels.

But not all killer T cells undergo apoptosis after battle. Some killer T cells that have lived long term become memory T cells. These cells already know how to easily recognize specific antigens, can easily amplify warning signals to the rest of the body, and contribute to combative immune response. How killer T cells become memory T cells is still unknown.

3. Regulatory T cells or Tregs live up to their name. They regulate the activity of T helper cells and even T memory cells by secreting cytokines or cell-to-cell contact (cell communication over a short distance). There are different types of Tregs, but the most efficient Tregs express different amounts of proteins CD4, CD25, and FOXP3 on their cell membranes.

4. Cytotoxic T cells have many other names such as CD8+ cells or killer T cells. Their only job is to eradicate any infected cell in the body, including cancerous cells. Cytotoxic T cells, when latching onto its target, will secrete several chemicals. It will release perforin to penetrate the infected cell’s membrane by creating tiny holes and release enzymes called granzymes and granulysin to kill the cell. Certain cytotoxic T cells can recognize abnormal proteins on the surface of cancer cells.

→ So how can we utilize T cells to fight cancer?

That’s where CAR T cell therapy comes in!

What is CAR T-cell therapy?

We are now aware that our own T cells possess the ability to fight cancer.

→ But what if we enhanced our T cells with bioengineering to speed up the act of doing so?

CAR T-cell therapy or chimeric antigen T-cell therapy, became the second gene therapy to be approved by the FDA in 2017 for treating children and young adults with leukemia. CAR T-cell therapy is a type of immunotherapy that introduces genetically-engineered T cells into the patient’s body using their own blood.

These new-and-improved T cells combine the best properties of B and T cells. For example, certain leukemia-affected cells express an antigen called CD19. Healthy B cells also express CD19 naturally and are prone to developing into leukemia cells. The new T cells will have receptors that bind to CD19 and also target cancer cells.

CAR T-cell therapy introduces a manmade receptor called the chimeric antigen receptor or CAR into a patient’s CD4 and CD8 T cells (remember Timmy?).

So what’s the process?

1. The patient’s blood sample must be properly separated by apheresis (separation of blood components), so the T cells can be properly extracted.

2) The hospital sends the blood sample to a centralized laboratory, where the remaining blood cells are modified.

3) Meanwhile, the patient must undergo chemotherapy to weaken their lymphocytes, which will ultimately improve the rate of proliferation and overall persistence of the modified T cells.

4) The activated T cells are infused back into the patient and ready to divide into more activated T cells to trigger apoptosis in only cancerous cells.

What makes up a CAR?

The CAR has three main components.

Source

The antigen recognition domain is the place where the antigen will bind to the receptor. In this case, CD19 will be that antigen.

Source

The spacer/hinge is responsible for tumor recognition, T cell proliferation, and T cell cytokine production (for warning signal amplification).

The intracellular signaling domain contains subdomains that also account for the T cell’s signaling processes in order to activate the unmodified T cells in the patient’s body.

Notably speaking, CAR T-cell therapy is a strong candidate for treating other forms of cancer for the near future.

Key Takeaways

1. T cells, a type of lymphoid cell, will start out as hematopoietic stem cells in the bone marrow.
2. T cells can differentiate based on random interaction of their CD8 and CD4 proteins with another cell’s MHC molecules.
3. Low affinity interactions > high affinity interactions.
4. A T cell must undergo apoptosis if it cannot pass the Double Negative Stage, Double Positive Stage, or any other stage in their development and training.
5. CAR T-cell therapy uses a patient’s own T cells in order to combat their cancer by equipping the T cells with a CAR.

Timmy and I want to thank you for taking the time to read this article!

I am a 16 year old high schooler who has a passion for learning about the immune system. I plan to expand my knowledge by writing more articles about other immunotherapies and cells involved in the immune system.

Do not hesitate to contact me at katelynwon@gmail.com or visit my LinkedIn.