Waterhouse et al. investigate the effects of deleting CTLA-4 from mice. These "CTLA-4 deficient" mice had disorders of T cell proliferation and quickly died.

Gribben et al. show that CTLA-4 functions to induce cell death of T cells. In biology, many signaling events are also controlled by an off switch that prevents prolonged or out-of-control signaling. CTLA-4 fulfills this role for T cells.

Brunet et al. identify the sequence of the CTLA-4 receptor. They note that it is part of immunoglobulin and has hydrophobic flanking sequences reminiscent of a membrane-bound protein. They also show that its expression is restricted to activated lymphocytes (T cells and B cells).

The Latin word ibidem means "in the same place". To save space, some authors use ibid. to refer to a reference from the same journal as the previous one.

Mueller, Jenkins, and Schwartz review the body of research on T cell costimulatory signaling pathways. In the few years after the discovery of the T-cell receptor, which directly binds the antigen to which a T cell is responding, researchers proposed that other signals are needed for T cells to become fully activated. This led to the two-signal model of T-cell activation described in this review.

Antigen-presenting cells. Specialized immune cells which allow T cells to be exposed to the antigens present in the body. This allows T cells to become activated so they can target those pathogens or diseased cells.

Within a living organism (as opposed to in vitro, or in cells grown in the lab).

Administering anti-CTLA-4 treatment in the absence of a tumor causes T cell responses to slow down. At the time this paper was published, it was not clear why this was the case.

The 2018 Nobel Prize in Physiology or Medicine was awarded jointly to James Allison and Tasuku Honjo for their work on antitumor immunity. Jim Allison is one of the authors on this seminal paper, in which the significant discoveries on the role of CTLA-4 were beginning to be made. Both James Allison and Tasuku Honjo showed that blocking inhibitory signals received by T cells can greatly enhance antitumor immunity. As you read in this paper, Jim Allison showed that blocking CTLA-4 achieves this effect; Tasuku Honjo was honored for similar work on another inhibitory molecule, PD-1. See the Nobel Prize announcement and an interview by a member of the Nobel committee here, and listen to various interviews with James Allison here.

By treating the mice with anti-CTLA-4, the authors were able to prevent tumor growth in two mice and delay it considerably in the other three.

The authors conducted a new set of experiments to check whether administering anti-CTLA-4 after tumors are detected is as effective as administering it at the same time tumors are introduced. If they are able to successfully treat mice after tumors are already established, then maybe this treatment could work for human patients as well!

The mice described in this section rejected the tumors twice: the first time, they were being treated with anti-CTLA-4, and the second time, they were not. This means that their immune system was able to fight off the second tumor on its own, and much faster than the immune systems of mice which had not previously survived exposure to the tumor. These are hallmarks of immunologic memory, the same phenomenon that ensures you only get chicken pox once in your life.

Unmodified: that is, wild-type, 51BLim10 cells do not have the extra genes which introduce B7 or the extra modifications which silence it.

The authors injected the mice which were previously treated with unmodified tumor. If they developed an immune memory, they may be able to clear this tumor even though it is not expressing B7 and they have not been given anti-CTLA-4 antibodies.

The authors repeated the experiment with an even lower tumor burden of 1x106 cells and found that the effectiveness of treatment was reduced at this dose. They do not propose a reason for this; perhaps you can think of one?

The mice were split into groups that received different treatment regimens. Two of the groups were untreated or were treated only with anti-CD28 antibodies.

The authors decided to check if there is an effect to changing the tumor dose. They halved the dose to 2x10(^6) and had a group of untreated and a group of anti-CTLA-4 treated mice.

This set of experiments was conducted with a variant of the same murine colon cancer tumor cells, but this time the tumors did not express B7. Thus the tumors are not able to provide the secondary signal to T cells.

The third group of mice was treated with antibodies targeting CTLA-4. Those mice were able to clear the tumors more quickly, by day 17, if they developed tumors at all.

The authors had a control group of mice which were injected with tumor cells expressing B7-1 molecules but were not treated with any antibodies.

The authors injected groups of mice with tumor cells expressing B7-1 molecules. The injections were administered within the membrane lining of the abdominal (peritoneal) cavity. These mice were then treated with two different regimens. One group of mice was injected with antibodies targeting CTLA-4 and another with antibodies targeting CD28.

The absence of CTLA-4 results in out-of-control cell division of T cells. Even though CTLA-4 is an inhibitory receptor, it is needed to produce the right balance of T cell activation, and its complete absence can be dangerous to organisms.

The antigen-binding fragment of antibody, i.e., the domain which binds specifically to the target of the antibody.

Using antibodies, scientists can induce dimerization of the receptor CTLA-4. Dimerization, a process whereby two similar molecules come together to form a single structure, is often the cause of signaling through a receptor. This experiment shows that the effect of CTLA-4 signaling is inhibition of T cell activation and expansion.

Latin for "in the glass." That is, experiments done in test tubes, petri dishes, flasks, or beakers, not in organisms.

Mueller, Jenkins, and Schwartz summarize the models proposed by several scientists to explain why T cells only respond to foreign invaders and not to peptide-MHC on healthy cells. They summarize it as the "two-signal model of T-cell activation," whereby there must be another signal necessary for T cells to be activated.

Science is collaborative.

Vision and Change Core Competency #5: Ability to communicate and collaborate with other disciplines. The Nature of Science and NGSS (#7): Science is a Human Endeavor.

A second exposure to the same threat. The immune system is, under certain conditions, able to remember threats it has encountered before and react to them more quickly and effectively upon each subsequent exposure.

Scientists often use models to represent complex systems. See NGSS Cross Cutting Concepts and Science and Engineering Practices (#2) and AP Science Practices (#1).

White blood cells central to adaptive immunity. T cells are able to recognize when cells are diseased and can kill them so they don't spread throughout the body.

The development of immune checkpoint inhibitor therapies for cancer won James Allison, one of the authors of this paper, and Tasuku Honjo the 2018 Nobel Prize in Physiology or Medicine. Read more at The Guardian.

There are many regulations on the use of animals in research studies. In 2019, these policies became even more stringent with the passing of the HEARTS Act of 2019. Read more here.

A laboratory strain of mice useful for studying cancer and immunology.

Krummel and Allison study the effect of CTLA-4 engagement. Their anti-CTLA-4 antibody blocks the receptor without engaging it. On its own, blocking the receptor does not have an effect on T-cell responses. Combined with an activation signal, anti-CTLA-4 allows for enhanced proliferation of T cells. The antibodies introduced here are the same ones used in the treatment of mice in this paper.

Walunas et al. show that CTLA-4 is an inhibitor of T cells. Their approach is to cross-link the receptors together, mimicking the cross-linking that would occur naturally upon receptor engagement. The effect of this is inhibition of T cell proliferation.

Linsley et al. study the differences between binding of CD28 and CTLA-4 to B7 molecules. Though they have similar avidities, they bind the B7 surface molecules using different conformations and kinetics.

Linsley et al. show that CTLA-4, like CD28, binds B7. Using a soluble version of CTLA-4, they show that it binds to B7 with an affinity of 12 nM.

Harper et al. present compelling evidence that CTLA-4 and CD28 share a similar function. They found that the receptors share similarities in structure, sequence, gene location, and expression patterns.

In this commentary, Linsley speculates on how the evidence for the roles of CD28 and CTLA-4 fits together. T-cell activation is more complex than scientists originally thought.

In this minireview, Bluestone summarizes the studies showing the nuances of CD28-mediated signaling. He discusses that different B7 molecules result in differing levels of activation, and that CTLA-4 has an inhibitory effect.

In this minireview, Jenkins summarizes the evidence for the role of CTLA-4. It was shown from multiple studies that CTLA-4 has an immunosuppressive effect.

Baskar et al. investigate the effect of B7 expression in tumor cells on the helper T cell immune response. They show that engineered tumor cells generate a helper T cell response and tumor rejection.

Townsend and Allison investigate whether expressing B7 on the surface of a tumor will enhance its rejection. They show that B7 expression results in a cytotoxic T cell immune response.

Chen et al. investigate whether expressing B7 on the surface of a tumor will enhance its rejection. They show a B7-dependent immune response by cytotoxic T cells.

June, Bluestone, Nadler, and Thompson review the research on B7 and CD28. Though they are the most commonly found members, each of these molecules is part of a larger receptor family of related molecules.

Linsley and Ledbetter summarize the research on CD28 and B7 molecules. The costimulatory signal needed for full T-cell activation is supplied through these surface molecules.

The process by which antigen-presenting cells uptake antigenic molecules from their surroundings, so that they may display them on their surface.

The process of exposing T cells to molecular signatures of disease through displaying them on the surface of antigen-presenting cells.

Outside of the living organism. In ex vivo experiments, cells originate in an organism, are extracted and modified, and then can be reintroduced.

The final tumor-specific method of generating immunity is to directly produce dendritic cells (the most common type of antigen-presenting cells) which display tumor antigens.

Another tumor-specific method of generating immunity is to modify tumor cells so that they produce a molecule which encourages the immune system's professional antigen-presenting cells (APCs) to take up molecules from the tumor cells. This means that APCs are better able to "train" T cells to recognize the signatures of the tumor.

Another tumor-specific method is modifying tumor cells so they become more similar to antigen-presenting cells. This allows T cells to recognize them more easily.

One such tumor-specific method of generating an immune response is to modify tumor cells so that they produce costimulatory molecules that enhance activation of T cells they come in contact with.

There are other methods of generating an immune response against cancer. Unlike this one, they are specific to the type of tumor, and require that the tumor cells be obtained and engineered in some way to become more visible to the immune system.

Anti-CTLA-4 treatment can be used for the normal tumors that a patient's immune system may get exposed to. It is not necessary to first extract tumor cells and modify them for the immune system to be able to recognize them. This makes it a very powerful treatment.

Another proposed mechanism of the effectiveness of CTLA-4 blockade is given. This mechanism could work in tandem with the previous one. Anti-CTLA-4 treatment may be allowing activated T cells to divide and grow for a longer period of time, so that there is a larger number of them circulating in the body and available to respond to the cancerous cells.

The authors propose that the effectiveness of CTLA-4 blockade may arise from lowering the number or concentration of antigens that T cells expect to be exposed to before they become fully activated. That is, anti-CTLA-4 may be allowing T cells to become more sensitive.

For the immune system to be able to respond to molecules on the surface of cancerous cells, these molecules are first presented on the surface of specialized immune cells. Through this antigen-presentation process, T cells able to respond to these molecules are activated and expanded.

Similar trends were observed in this experiment. Mice left untreated grew tumors rapidly, while the majority of those treated with anti-CTLA-4 remained tumor-free.

The experiment was repeated with a much larger number of tumor cells injected. This is to ensure that the effects seen rely only on the treatment course and not on other factors.

Those mice that were not treated grew tumors in the first week of the experiment.

The authors injected groups of 5 mice each with SA1N cells causing fibrocarcinoma.

Another laboratory strain of mice useful for studying cancer and immunology.

Not only was delaying treatment effective, it was more effective than treating the mice on the same day that tumors are introduced.

Most of the previously immunized mice did not develop tumors of detectable size; that is, their immune system was able to kill all the tumor cells almost as soon as they were introduced. Only one of the mice developed a detectable tumor, but the growth was very slow.

If mice were not previously exposed to the tumor cells along with a treatment, they did not fare very well.

The mice which previously mounted an immune response with the help of anti-CTLA-4 were able to leverage a similar response toward this re-exposure. Remember that naive mice exposed to half this many tumor cells were not able to control the cancer's growth.

The treated mice had much better outcomes than the untreated mice, which were euthanized after 35 days.

The mice treated with antibodies against CTLA-4 completely recovered and were able to clear the tumors.

A third group of mice received anti-CTLA-4 antibodies as treatment.

The mice which received these treatments could not control the growth of their tumors. They died 35 days after the tumors were injected.

Two groups were injected with anti-CD28 or not treated. Most of the mice in these two groups cleared the tumors, but did so by day 23. Two of the mice did not completely clear the tumors but were able to keep them from growing further.

If cancerous cells express B7-1, the immune system is better able to respond to them and clear the tumor.

Relating to or originating from mice.

An antibody able to bind two of its targets at once.

The pieces of evidence outlined in this paragraph led the authors to conclude that blocking tumors from being able to engage CTLA-4 might result in T cells being better able to fight off tumors.

Another effect of CTLA-4 signaling is the programmed cell death of T cells. This is another way for CTLA-4 to dampen T cell responses.

Blocking CTLA-4 also increases T cell activation that happens as a result of encountering foreign invaders.

The inhibitory CTLA-4 receptor can be blocked by using soluble antibody. Blocking the effects of CTLA-4 allows T cells to activate and proliferate normally upon induction by anti-CD3.

CD3 is a co-receptor alongside the T-cell receptor. Together, they provide the primary signal of T cell activation. In this experiment, the scientists tried to add anti-CD3 antibody, which would normally result in T cell activation. However, the inhibitory effect of CTLA-4 wins out over this activation technique.

Two indicators of T cell activation. Once activated, T cells divide rapidly and produce a molecule called interleukin-2.

CTLA-4 competes with CD28 for binding to the B7 family of molecules.

A related protein, usually with very similar sequence and structure.

The overall activation of T cells is also influenced by another type of secondary signal. In addition to stimulatory signals discussed above, T cells may also encounter inhibitory signals. The balance of these two signaling events results in the overall level of activation.

Introducing the genes for B7 molecules into tumor cells allows them to provide the secondary signal needed to activate T cells. This allows the immune system to respond to those modified tumor cells, as well as any future tumors of the same type that they encounter (even if the latter do not express B7).

A technique by which the genes in a cell are modified.

The two-signal model of T-cell activation was confirmed when scientists found that specialized immune cells, called antigen-presenting cells, provide a secondary signal to T cells in order to activate them. This secondary signal comes from the interaction of the B7 family of molecules (on the surface of APCs) with the CD28 receptor (on the surface of T cells).

A family of binders to CD28. The two most important members are B7-1 and B7-2, mentioned below.

The target for T cell receptors is always a short peptide displayed on the surface of the cells. The protein responsible for displaying the peptides is called the major histocompatibility complex.

The receptor used by T cells to recognize specific antigens.

Molecules recognized by the immune system; signatures of disease.

Administering antibodies against CTLA-4 also allows the mice to prevent establishment of tumors due to future exposure.

By preventing CTLA-4 from binding to its ligands, T cells can now be activated more potently. This allows them to kill cancer cells.

The authors injected mice with antibodies that bind CTLA-4.

Another receptor on the surface of T cells, with an opposite effect compared to CD28. Binding to CTLA-4 causes damping of T cell activation.

To become fully activated, T cells need to receive a signal through the CD28 receptor on their surface.

The ability of the immune system to recognize diseased or foreign cells.