CD4: The Road to Redemption

Dear Internet,

You may have heard of helper T cells, also called CD4+ T cells. These T cells have a lot of parts to them, but I want to tell you about CD4 – the surface protein that gives these cells their name. Why should you want to continue reading about this protein? Well I’ll give you a teaser to the end of this post – CD4 has potential to protect humans from HIV – a retrovirus you may have heard of that currently affects over 1.2 million people in the United States (aids.gov).

Umm yes please. You probably want to keep reading.

Part One: Integral in immune response

CD4 is a surface protein on immune cells. It spans the cell membrane and has both an extracellular and intracellular domain.

The four extracellular domains of CD4. Image derived from PDB structure 1WIO (DOI: 10.2210/pdb1wio/pdb) using pyMOL (Wu, Kwong, & Hendrickson, 1997).

The extracellular portion of CD4 has 4 immunoglobulin or antibody domains, labelled D1 to D4. This extracellular domain is key and something each of us should be thankful for in cold and flu season. This is because this domain binds to the MHC II molecule located on antigen presenting cells (APCs). Antigen presenting cells are cells which internalize foreign antigens and then present them to T cells. T cells cannot bind to free foreign antigens – they need them to be presented to them. The T cell’s important T cell receptor (TCR) binds to the APC, but importantly CD4 is a coreceptor that also binds to the MHC II molecule on APCs.  It is because of CD4 and TCR that T cells are activated and an immune response begins (Vignali et al., 1993).

So that was the extracellular domain of CD4. What about the intraceullar part? Well it is important in the signaling cascade involved with T cells. When antigen and TCR are bound, TCR complexes form. At this point, the cytoplasmic tail of CD4 interacts with Lck, a protein tyrosine kinase.  Lck triggers a tyrosine phosphorylation signaling cascade which eventually leads to activation of transcription factors (Janeway et al., 2005).

Interactions between CD4 and the TCR, MHC class II and Lck (Vignali et al., 1993).

So CD4 is involved as a receptor and in signal transduction in T cells. Pretty important. You can see why the CD4 t cells use it in their name! Check out the black and white photo above – it illustrates all we’ve seen so far well. You’ll see Lck represented as p56lck.

If you have had any experience with HIV or AIDS, you will have heard of CD4 cell counts. What happens in HIV infection is that the HIV retrovirus uses CD4 as its host cell to make copies of itself. In the process, it destroys the CD4 T cells. HIV can in time destroy enough of the body’s CD4 cells so that it can’t carry out immune responses. This is the point where HIV leads to AIDS, the final stage of HIV infection. For this reason, measuring CD4 cell counts is an important measurement taken by people infected with HIV to monitor the strength of their immune system (aids.gov).

CD4 cell counts are done using a blood sample that is analyzed using a CD4 antibody which is attached to some sort of fluorescent or other easily detected tag. Why do I tell you this? Well if declining CD4 cell levels are indicative of badly damaged immune systems and leads to infections; this clearly indicates the importance of CD4 and CD4 cells in our bodies.

Part Two: I’m not impressed. How is this going to protect me from HIV?  
“But Susan,” you may be thinking, “CD4 actually sounds pretty lame. CD4 is just an easy target of HIV. Then once its destroyed, it causes major problems for our immune systems! No way is this protein worthy of being protein of the year.”

I will admit this is disheartening – until we see how the CD4 protein itself can be used to halt this. You see, this is a protein on a route to redemption. And that is why it should be the 2015 Protein of the year.

So I mentioned that CD4 T cells acts as the host cell for HIV. It turns out that the CD4 protein is the surface protein on these cells that HIV first binds to. It is the access point for this virus.

HIV’s Env (envelope) protein binds first to CD4, after which it undergoes conformational changes that allow it to bind to a second cellular receptor called CCR5. This then leads to fusion between Env and the T cell membrane, allowing infection of HIV (Wilen et al., 2012).

You might think that this isn't looking much better for CD4’s case. The thing is, because of its role as the first receptor on T cells, CD4 protein can be used as a decoy to fake out HIV. In a recent Nature article from March 2015, the Farzan research group reports that eCD4Ig – a derivative of CD4 –acts as an HIV entry inhibitor that if delivered by the gene-therapy vector AAV, can act as an effective alternative to conventional vaccines for HIV (!!!). The Farzan group created the artificial antibody eCD4Ig by taking CD4 and attaching the Fc domain of the human Immunoglobulin G antibody, as well as a small sulfopeptide mimic of the second cellular receptor, CCR5  (Gardner et al., 2015).

“CD4-Ig is comprised of CD4 domains 1 and 2 (blue) fused to the human IgG1 Fc domain (grey). In eCD4-Ig, the sulfopeptide CCR5mim1 (red) is fused to the C terminus of CD4-Ig. The sequence of the CCR5 N terminus is provided for comparison” (Gardner, et al., 2015).

The HIV Env binds to this eCD4Ig instead of the normal CD4 on a T cell. This eCD4Ig binding irreversibly inhibits Env, thus preventing HIV infection (Gardner et al., 2015). The image below shows a model of how eCD4Ig binds to the Env trimer.

A model of how eCD4-Ig may associate with Env. The purple regions are two of the three Env trimers. The IgG1 Fc domain of eCD4-Ig is shown in cyan. The red region represents one of the four extracellular domains of CD4. The sulfopeptide mimics of CCR5 are shown as green, yellow and red spheres. (Gardner, et al., 2015).

eCD4Ig was seen to outperform current bNAbs (broadly neutralizing antibodies) in terms of potency and breadth. After seeing eCD4Ig outperform other HIV antibodies in cell culture studies, the Farzan group then introduced eCD4Ig in rhesus macaques, a species of monkey, through the harmless AAV virus. The macaques were given increasing doses of SHIV (Simian-Human Immunodeficiency Virus) for 34 weeks, but none of the e-CD4Ig macaques were infected, while all the non-eCD4Ig macaques were. In the words of the article, “Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine” (Gardner et al., 2015).

So CD4, while you are right at the heart of many potential problems with the immune system, we now see how YOU, one little protein, could potentially fix this whole problem. All you need are a few tweaks and a more complicated initialism. Your redemption story is inspiring. That is why you deserve to be the 2015 Protein of the Year. Here’s to hoping you can turn into the amazing HIV vaccine we all believe you can be.

References:

Gardner, M. R., Kattenhorn, L. M., Kondur, H. R., von Schaewen, M., Dorfman, T., Chiang, J. J., … Farzan, M. (2015). AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges. Nature, 519(7541), 87–91. doi:10.1038/nature14264

Janeway CA, Travers P, Walport M, Shlomchik MJ. 2005. ImmunoBiology, Sixth Edition. New York: Garland Science Taylor and Francis Group.

Vignali, D. A., Doyle, C., Kinch, M. S., Shin, J., & Strominger, J. L. (1993). Interactions of CD4 with MHC class II molecules, T cell receptors and p56lck. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, 342(1299), 13–24. http://doi.org/10.1098/rstb.1993.0130

Wilen, C. B., Tilton, J. C., & Doms, R. W. (2012). HIV: cell binding and entry. Cold Spring Harbor Perspectives in Medicine, 2(8). http://doi.org/10.1101/cshperspect.a006866

"What Is HIV/AIDS?" Aids.gov. U.S. Department of Health & Human Services., 29 Apr. 2014. Web. 11 Mar. 2015. <https://www.aids.gov/hiv-aids-basics/hiv-aids-101/what-is-hiv-aids/index.html>.

Wu, H., Kwong, P. D., & Hendrickson, W. A. (1997). Dimeric association and segmental variability in the structure of human CD4. Nature, 387(6632), 527–530. http://doi.org/10.1038/387527a0

CD4: The Road to Redemption

Dear Internet,

You may have heard of helper T cells, also called CD4+ T cells. These T cells have a lot of parts to them, but I want to tell you about CD4 – the surface protein that gives these cells their name. Why should you want to continue reading about this protein? Well I’ll give you a teaser to the end of this post – CD4 has potential to protect humans from HIV – a retrovirus you may have heard of that currently affects over 1.2 million people in the United States (aids.gov).

Umm hell yes. You probably want to keep reading.

Part One: Integral in immune response

CD4 is a surface protein on immune cells. It spans the cell membrane and has both an extracellular and intracellular domain.

The four extracellular domains of CD4. Image derived from PDB structure 1WIO (DOI: 10.2210/pdb1wio/pdb) using pyMOL (Wu, Kwong, & Hendrickson, 1997).

The extracellular portion of CD4 has 4 immunoglobulin or antibody domains, labelled D1 to D4. This extracellular domain is key and something each of us should be thankful for in cold and flu season. This is because this domain binds to the MHC II molecule located on antigen presenting cells (APCs). Antigen presenting cells are cells which internalize foreign antigens and then present them to T cells. T cells cannot bind to free foreign antigens – they need them to be presented to them. The T cell’s important T cell receptor (TCR) binds to the APC, but importantly CD4 is a coreceptor that also binds to the MHC II molecule on APCs.  It is because of CD4 and TCR that T cells are activated and an immune response begins (Vignali et al., 1993).

So that was the extracellular domain of CD4. What about the intraceullar part? Well it is important in the signaling cascade involved with T cells. When antigen and TCR are bound, TCR complexes form. At this point, the cytoplasmic tail of CD4 interacts with Lck, a protein tyrosine kinase.  Lck triggers a tyrosine phosphorylation signaling cascade which eventually leads to activation of transcription factors (Janeway et al., 2005).

Interactions between CD4 and the TCR, MHC class II and Lck (Vignali et al., 1993).

So CD4 is involved as a receptor and in signal transduction in T cells. Pretty important. You can see why the CD4 t cells use it in their name! Check out the black and white photo above – it illustrates all we’ve seen so far well. You’ll see Lck represented as p56lck.

If you have had any experience with HIV or AIDS, you will have heard of CD4 cell counts. What happens in HIV infection is that the HIV retrovirus uses CD4 as its host cell to make copies of itself. In the process, it destroys the CD4 T cells. HIV can in time destroy enough of the body’s CD4 cells so that it can’t carry out immune responses. This is the point where HIV leads to AIDS, the final stage of HIV infection. For this reason, measuring CD4 cell counts is an important measurement taken by people infected with HIV to monitor the strength of their immune system (aids.gov).

CD4 cell counts are done using a blood sample that is analyzed using a CD4 antibody which is attached to some sort of fluorescent or other easily detected tag. Why do I tell you this? Well if declining CD4 cell levels are indicative of badly damaged immune systems and leads to infections; this clearly indicates the importance of CD4 and CD4 cells in our bodies.

Part Two: I’m not impressed. How is this going to protect me from HIV?  

“But Susan,” you may be thinking, “CD4 actually sounds pretty lame. CD4 is just an easy target of HIV. Then once its destroyed, it causes major problems for our immune systems! No way is this protein worthy of being protein of the year.”

I will admit this is disheartening – until we see how the CD4 protein itself can be used to halt this. You see, this is a protein on a route to redemption. And that is why it should be the 2015 Protein of the year.

So I mentioned that CD4 T cells acts as the host cell for HIV. It turns out that the CD4 protein is the surface protein on these cells that HIV first binds to. It is the access point for this virus.

HIV’s Env (envelope) protein binds first to CD4, after which it undergoes conformational changes that allow it to bind to a second cellular receptor called CCR5. This then leads to fusion between Env and the T cell membrane, allowing infection of HIV (Wilen et al., 2012).

You might think that this isn’t looking much better for CD4’s case. The thing is, because of its role as the first receptor on T cells, CD4 protein can be used as a decoy to fake out HIV. In a recent Nature article from March 2015, the Farzan research group reports that eCD4Ig – a derivative of CD4 –acts as an HIV entry inhibitor that if delivered by the gene-therapy vector AAV, can act as an effective alternative to conventional vaccines for HIV (!!!). The Farzan group created the artificial antibody eCD4Ig by taking CD4 and attaching the Fc domain of the human Immunoglobulin G antibody, as well as a small sulfopeptide mimick of the second cellular receptor, CCR5.

“CD4-Ig is comprised of CD4 domains 1 and 2 (blue) fused to the human IgG1 Fc domain (grey). In eCD4-Ig, the sulfopeptide CCR5mim1 (red) is fused to the C terminus of CD4-Ig. The sequence of the CCR5 N terminus is provided for comparison” (Gardner, et al., 2015).

The HIV Env binds to this eCD4Ig instead of the normal CD4 on a T cell. This eCD4Ig binding irreversibly inhibits Env, thus preventing HIV infection (Gardner et al., 2015). The image below shows a model of how eCD4Ig binds to the Env trimer.

A model of how eCD4-Ig may associate with Env. The purple regions are two of the three Env trimers. The IgG1 Fc domain of eCD4-Ig is shown in cyan. The red region represents one of the four extracellular domains of CD4. The sulfopeptide mimicks of CCR5 are shown as green, yellow and red spheres. (Gardner, et al., 2015).

eCD4Ig was seen to outperform current bNAbs (broadly neutralizing antibodies) in terms of potency and breadth. After seeing eCD4Ig outperform other HIV antibodies in cell culture studies, the Farzan group then introduced eCD4Ig in rhesus macaques, a species of monkey, through the harmless AAV virus. The macaques were given increasing doses of SHIV (Simian-Human Immunodeficiency Virus) for 34 weeks, but none of the e-CD4Ig macaques were infected, while all the non-eCD4Ig macaques were. In the words of the article, “Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine” (Gardner et al., 2015).

So CD4, while you are right at the heart of many potential problems with the immune system, we now see how YOU, one little protein, could potentially fix this whole problem. All you need are a few tweaks and a more complicated initialism. Your redemption story is inspiring. That is why you deserve to be the 2015 Protein of the Year. Here’s to hoping you can turn into the amazing HIV vaccine we all believe you can be.

References:

Gardner, M. R., Kattenhorn, L. M., Kondur, H. R., von Schaewen, M., Dorfman, T., Chiang, J. J., … Farzan, M. (2015). AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges. Nature, 519(7541), 87–91. doi:10.1038/nature14264

Janeway CA, Travers P, Walport M, Shlomchik MJ. 2005. ImmunoBiology, Sixth Edition. New York: Garland Science Taylor and Francis Group.

Vignali, D. A., Doyle, C., Kinch, M. S., Shin, J., & Strominger, J. L. (1993). Interactions of CD4 with MHC class II molecules, T cell receptors and p56lck. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences, 342(1299), 13–24. http://doi.org/10.1098/rstb.1993.0130

Wilen, C. B., Tilton, J. C., & Doms, R. W. (2012). HIV: cell binding and entry. Cold Spring Harbor Perspectives in Medicine, 2(8). http://doi.org/10.1101/cshperspect.a006866

"What Is HIV/AIDS?" Aids.gov. U.S. Department of Health & Human Services., 29 Apr. 2014. Web. 11 Mar. 2015. <https://www.aids.gov/hiv-aids-basics/hiv-aids-101/what-is-hiv-aids/index.html>.

Wu, H., Kwong, P. D., & Hendrickson, W. A. (1997). Dimeric association and segmental variability in the structure of human CD4. Nature, 387(6632), 527–530. http://doi.org/10.1038/387527a0

A CD4 derivative prevents SHIV infection in monkeys!

CD4 caught my interest because not only is it essential in the immune system of the human body, but it is also an important component in the infection of HIV. Because of this, there has been recent research of how CD4 derivatives could be used in HIV treatment. This protein seems to be at the center of a lot, so I wanted to investigate it further.

Interactions of CD4 with MHC class II molecules, T cell receptors and p56Lck

Vignali, D.A., Doyle, C., Kinch, M.S., Shin, J. & Strominger, J.L. Interactions of CD4 with MHC class II molecules, T cell receptors and p56Lck. Phil. Trans.: Biol. Sciences 342, 13–24 (1993).

There have been many studies on many aspects of the CD4 protein, so a review article is a necessary starting point. First, it provides some necessary background info.

CD4 is a coreceptor located on T Cells. It is a coreceptor along with the TCR (T cell receptor). Together the TCR and CD4 recognize antigens presented by antigen-presenting cells. T Cells that have the CD4 coreceptor on their surface are called CD4 T Cells. These CD4 T cells bind specifically to MHC class II molecules which are located on antigen presenting cells. In contrast, a different coreceptor CD8 binds to MHC class I molecules.

These CD4 T cells are called “helper” T cells because once the antigen is recognized, the T cell is activated and sends out many cytokine proteins. There are many different kinds of cytokines that all facilitate different types of immune responses.

Image source: Vignali, D.A., Doyle, C., Kinch, M.S., Shin, J. & Strominger, J.L. Interactions of CD4 with MHC class II molecules, T cell receptors and p56Lck. Phil. Trans.: Biol. Sciences 342, 13–24 (1993).

This review paper from 1993 is old, but provides a good introduction to the basic function and actions of CD4. The review discusses three important interactions of CD4:

1) investigations showing that CD4 binds with MHC II on the surface of an APC 

2) how CD4 physically associates with the TCR, leading to T cell activation 

3) how phosphorylation regulates and affects interaction between CD4 and the protein tyrosine kinase p56lck, also located on the Tcell.

HIV: Cell binding and entry

Wilen C.B., Tilton J.C., Doms R.W. HIV: Cell binding and entry. Cold Spring Harb. Perspect. Med. 2012;2 doi: 10.1101/cshperspect.a006866

Image source: Wilen C.B., Tilton J.C., Doms R.W. HIV: Cell binding and entry. Cold Spring Harb. Perspect. Med.2012;2 doi: 10.1101/cshperspect.a006866

Next, a review article about HIV and its infection sheds light on the significance of CD4 in disease. The first step of the HIV replication cycle is to bind and enter into the host cell. CD4 serves as a host protein that HIV’s Env (envelope) protein binds to. After binding to CD4,  Env undergoes conformational changes that allow it to bind to the second cellular receptor CCR5. This then leads to membrane fusion, allowing infection of HIV. The review article also provides information on the history of discovery of CD4 and HIV binding as well as info into how CD4 signalling from p56lck effects effective HIV infection.

AAV-expressed eCD$-Ig provides durable protetion from multiple SHIV challenges.

Gardner, M. R., Kattenhorn, L. M., Kondur, H. R., von Schaewen, M., Dorfman, T., Chiang, J. J., … Farzan, M. (2015). AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges. Nature, 519(7541), 87–91. doi:10.1038/nature14264

Image source: https://www.flickr.com/photos/jimnicholson/6005070459/

This Nature article is what attracted me to CD4 in the first place: Monkeys who are immune to SHIV. Too good to be true? Let's see.

This is a very recent primary research article from March 2015. In the study, the Farzan research group’s goal is to attempt to create an entry inhibitor of HIV that is more efficient than current bNAbs (broadly neutralizing antibodies) which require high concentration for protection. The Farzan group created an artificial antibody by taking CD4 and attaching a small piece of the second cellular receptor, CCR5. This new construct was named e-CD4Ig. The idea is that HIV will bind to this eCD4Ig instead of a CD4 on a T cell, thus preventing infection.

The Farzan group saw that eCD4Ig outperformed other HIV antibodies in cell culture studies. E-CD4Ig  then was introduced in rhesus macaques, a species of monkey, by inserting it in the harmless AAV virus which was given to the macaques. The macaques were given increasing doses of SHIV (SHIV stands for Simian-Human Immunodeficiency Virus) for 34 weeks, but none of the e-CD4Ig macaques were infected, while non e-CD4Ig macaques were. In the words of the article, “Our datasuggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.”

CD4 - first look

CD4 is a glycoprotein on the surface of T-cells in the human body. It is has four immunoglobulin or antibody domains, labelled D1 to D4.  CD4 is of particular interest because HIV binds to CD4 on a T-cell facilitating HIV infection.

The 1CDH structure on the Protein Database shows a crystallization of domains D1 and D2 of CD4. The D1 and D2 amino-terminal domains contain the residues involved in HIV interaction.

CD4 consists primarily of beta sheets (red) connected by loops (pink). There are two small helices in the D1 and D2 domains (light blue).

Below is a model showing the surface of CD4 D1 and D2. 

There are two disulfide bonds within the D1 and D2 domains between Cys16&Cys84 and Cys130&Cys159 (shown in red).

The image below shows a spacefilling model of the protein using vanderWaal's radii. Each sphere is color labelled by atom: Carbon (green), Nitrogen (blue), Oxygen (red).

All images derived from PDB structure 1CDH (DOI: 10.2210/pdb1cdh/pdb) using pyMOL.