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The Chemistry of Immune Activation: the redox environment in T cell activation and HIV infection

July 10, 2011

500 Fig1 T cell redox

ResearchBlogging.org

My PI recently forwarded an article to me from the NIH glycoimmunology listserv“Galectin-9 binding to cell surface protein disulfide isomerase regulates the redox environment to enhance T-cell migration and HIV entry” by Shuguang Bi et al. – which discusses the effects of galectin-9 on T cell activity and infectability. Galectins are sugar-binding proteins, expressed by most cells of the immune system, involved in immune regulation and pathogen recognition (via surface glycans)(1). Being sugar-binding proteins, galectins will bind viral glycoproteins, sometimes inhibiting viral fusion/entry, as is the case for galectin-1 interaction with Nipah virus (1). However, Bi et al. found that galectin-9 has the opposite effect during HIV infection, promoting HIV entry via its interaction with protein disulfide isomerase (PDI) at the surface of T cells (2). HIV is not the only virus that takes advantage of host galectin expression. Although the role of galectins in antiviral immunity is not yet clearly defined, the alteration of galectin expression levels and function during viral infection implies a role for these sugar-binding proteins at multiple stages of the antiviral immune response (1).

Although this paper touches on an interesting topic in antiviral immunity, it is more of a biochemistry paper than an immunology paper, focusing more on the redox environment during galectin-9 interaction with T cells. This makes it a challenging read for someone who doesn’t have a very strong background in biochemistry. However, this paper showed me that biochemistry can teach us a lot about immune function during infection. In acknowledgement of this, I am starting this post with a review of the biochemistry terms and information needed as a background for understanding the Bi et al. data and its relevance.

Disulfide bonds and oxidoreductases

The thought of disulfide bonds gives me flashbacks from Biochem 101, conjuring up images of 3D protein structures – twists of beta-sheets and alpha-helices brought together to form functional globules. These functional protein forms/globules are mutable, and they are often rearranged via the activity of enzymes called oxidoreductases, or more specifically, thiol isomerases. Oxidoreductases are enzymes that catalyze the transfer of electrons from one molecule to another. Thiol isomerases are oxidoreductases that act on sulfur groups, and are capable of oxidizing (forming), reducing (breaking apart), and rearranging disulfide bonds (3). Although traditionally believed to be located exclusively in the endoplasmic reticulum, where they assist in protein folding; more recent studies show that these enzymes can also be found at the cell surface, where their activities have different functional consequences (4). How they get to the cell surface is still a subject of debate. However, there are now some excellent examples in the literature of how thiol isomerases function at the cell surface to regulate the redox environment and immune activation.

The redox environment and immune activation:

B cells and T cells require a reducing environment for their growth and activation, respectively (5). Biochemically speaking, this means that most of the amino acid cysteine should be in the free thiol form (cysteine) as opposed to the disulfide-bonded form (cystine). In the case of T cells, free cysteine is required for synthesis of glutathione, necessary for proliferation of these cells (5). Although a reducing environment is preferred for immune activation; for most tissues, the extracellular environment is more oxidizing. An exception to this occurs at the lymph nodes, where a slightly more reduced environment persists (5). Luckily, antigen presenting cells are able to assist in generating and maintaining a more reducing environment (Fig. 1). Upon encountering antigen, dendritic cells secrete glutathione, which is cleaved to become cysteine/free thiols in the extracellular space (6). Additionally, when the dendritic cell encounters and interacts with an antigen-specific T cell, it secretes the oxidoreductase thioredoxin (TRX), which further contributes to the amount of thiols in the extracellular space that are available for T cells to take in (7). Dendritic cells are not the only antigen-presenting cells capable of creating a reducing environment for initiation of an adaptive immune response. Monocytes, macrophages and B cells have also been shown to release free thiols consistent with their roles in antigen presentation (5).

Protein Disulfide Isomerases:

Like the TRX example above, immune activation can also result in increased amounts of protein disulfide isomerase (PDI) on the surface of immune cells. Here it may interact with various protein receptors, making and breaking disulfide bonds, altering the extracellular redox environment and cellular function (4). PDI’s presence has been demonstrated on the surface of platelets, B cells, and T cells, but its activity on the surface of platelets has been the most studied (4).

Platelets are subcellular fragments present in the blood that respond to vascular injury by forming a plug, stopping blood flow out of the damaged blood vessel. Plug formation (also known as primary hemostasis) consists of a series of three events (8). First, the individual platelets adhere to the injured/damaged blood vessel wall (8). Adherent platelets then release their granule contents, freeing various platelet-activating factors (8). Finally, the activated adjacent platelets adhere to each other (8). This final interaction is mediated by the platelet integrin aIIbb3, which links platelets together via fibrinogen (check out Fig. 1 in Medscape article) (8). Platelet aIIbb3 contains many cysteine residues, and its binding activity is controlled by their arrangement (8). During platelet activation, surface PDI catalyzes an event which rearranges aIIbb3’s disulfide bonds, converting it from the inactive to the active fibrinogen-binding form (8). In this manner, PDI is essential for platelet adhesion, and primary hemostasis.

The role of Galectin-9 in retaining PDI at the cell surface:

Although several studies confirm PDI’s presence on the surface of immune cells, it wasn’t until last month (June 2011) that a study explained how PDI is retained at the cell surface. As part of a search for galectin-9 receptors on the surface of T cells, S. Bi et al. discovered that galectin-9 is a PDI ligand. Since galectins are generally known to bind glycoprotein receptors, retaining them at the cell surface, the group considered that galectin-9 could be involved in retaining PDI at the surface of T cells. To test this, they added galectin-9 to T cell culture and quantified surface PDI by flow. Surface PDI expression increased on the surface of PhaRST6 cells (murine T cell line resistant to galectin-9-mediated cell death) and murine Th2 cells, peaking at 2 hours post-addition of galectin-9. As PDI RNA was not increased by galectin-9 addition, it was assumed that the increased amount of PDI at the T cell surface was due to retention and not de novo synthesis. Increased surface PDI expression was accompanied by increased cell surface thiols, suggesting thioreductase activity, and a role for galectin-9 in regulating the redox environment on the surface of T cells via interaction with PDI.

640 gal9 fig2

Galectin-9, the redox environment, and T cell migration:

The effects of redox status on T cell activation have already been described both in the literature and in this blog post. Since redox status can also affect leukocyte migration (2), Bi et al. assayed for differences in T cell migration in response to galectin-9 using a Matrigel migration assay. Migration of Th2 cells, which highly express PDI at their surface, was increased upon addition of galectin-9. This migration is PDI-dependent, as it was blocked by addition of an anti-PDI antibody. Th1 cells, which express very little PDI, died at the surface of the Matrigel instead of migrating through it (Fig. 2).

In addition to galectin-9, Bi et al also found PDI associated with CD61 (also known as b3 integrin – a subunit of the platelet fibrinogen receptor aIIbb3 discussed above) on the surface of PhaRST6 and primary Th2 cells. Antibody-mediated neutralization of CD61 showed that it also contributes to increased T cell migration observed upon galectin-9 addition, most likely by its association with PDI at the T cell surface.

The Bi et al. study added a lot to our collective knowledge of how thiol isomerases and the redox environment affect T cell function. Prior to this study, it was known that a reducing environment is required for T cell activation, and that thiol isomerases contribute to establishing/maintaining this environment. Now it is known that galectin-9 also affects the redox environment by retaining the thiol isomerase PDI at the T cell surface and specifically encouraging migration of Th2 cells, which highly express PDI and CD61 at their surface. The differential effects of galectin-9 on Th1 and Th2 cells presented in the Bi et al. publication are intriguing, and this group’s work suggests that in addition to affecting T cell activation, the redox environment may also affect which subset of T helper cells (Th1 or Th2) are activated (see the biolegend Th1 Th2 guide for more on Th1 and Th2 development).

PDI and HIV entry:

Perhaps even more intriguing, for an HIV researcher like myself, is evidence showing that thiol reductases can interact with proteins involved in HIV infection (9). PDI associates with the HIV receptor CD4 (9). When HIV binds to CD4 on the surface of T cells, PDI cleaves disulfide bonds in viral gp120, facilitating viral entry (9). When PDI is inhibited, HIV cannot enter T cells, likely due to inhibition of virus-cell fusion (9). Consistent with this, increasing surface PDI levels by adding galectin-9 to T cell cultures results in increased HIV infection (2). Unfortunately, what these data tell us is that certain proteins required for T cell activation – thioredoxin and PDI – facilitate HIV infection. This appears to be just one more way in which HIV outsmarts our immune system, utilizing immune activation to gain entry into T cells. However, the good news is that we may be able to counteract this activity using PDI inhibitors in vivo (9). Although a PDI inhibitor would likely inhibit T cell activation to a certain extent, immune activation has been shown to be generally disadvantageous in HIV patients.

In designing novel HIV immunotherapeutics, controlling chronic immune activation is rapidly becoming a priority. The studies I described in this post suggest that we should consider if and how novel immunotherapeutic glycoproteins will interact with HIV receptors/coreceptors, as proteins bound to these receptors may affect receptor conformation and directly interact with HIV during its entry. Additionally, they show that alterations to the redox environment should be considered in HIV drug/therapy development, as the redox environment has proven effects on both the immune response and viral entry.

Citations:

(1) Vasta, G. (2009). Roles of galectins in infection Nature Reviews Microbiology, 7 (6), 424-438 DOI: 10.1038/nrmicro2146

(2) Bi S, Hong PW, Lee B, & Baum LG (2011). Galectin-9 binding to cell surface protein disulfide isomerase regulates the redox environment to enhance T-cell migration and HIV entry. Proceedings of the National Academy of Sciences of the United States of America, 108 (26), 10650-5 PMID: 21670307

(3) Jordan, P., & Gibbins, J. (2006). Extracellular Disulfide Exchange and the Regulation of Cellular Function Antioxidants & Redox Signaling, 8 (3-4), 312-324 DOI: 10.1089/ars.2006.8.312

(4) Turano, C., Coppari, S., Altieri, F., & Ferraro, A. (2002). Proteins of the PDI family: Unpredicted non-ER locations and functions Journal of Cellular Physiology, 193 (2), 154-163 DOI: 10.1002/jcp.10172

(5) Castellani, P., Angelini, G., Delfino, L., Matucci, A., & Rubartelli, A. (2008). The thiol redox state of lymphoid organs is modified by immunization: Role of different immune cell populations European Journal of Immunology, 38 (9), 2419-2425 DOI: 10.1002/eji.200838439

(6) Yan, Z., Garg, S., Kipnis, J., & Banerjee, R. (2009). Extracellular redox modulation by regulatory T cells Nature Chemical Biology, 5 (10), 721-723 DOI: 10.1038/nchembio.212

(7) Angelini, G. (2002). From the Cover: Antigen-presenting dendritic cells provide the reducing extracellular microenvironment required for T lymphocyte activation Proceedings of the National Academy of Sciences, 99 (3), 1491-1496 DOI: 10.1073/pnas.022630299

(8) Essex, D. (2004). The Role of Thiols and Disulfides in Platelet Function Antioxidants Redox Signaling, 6 (4), 736-746 DOI: 10.1089/1523086041361622

(9) Matthias, L., & Hogg, P. (2003). Redox Control on the Cell Surface: Implications for HIV-1 Entry Antioxidants & Redox Signaling, 5 (1), 133-138 DOI: 10.1089/152308603321223621

2 Comments leave one →
  1. agnes-laurence chenine permalink
    July 12, 2011 7:05 AM

    very interesting. I keep learning a lot reading your blog.

  2. July 27, 2011 8:50 AM

    Enlightening as always, thanks!

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