The Role of CD40–CD154 Interactions in Autoimmunity and the Benefit of Disrupting this Pathway

Autoimmune disease is associated with adaptive immune component dysfunction such as the B cells and T cells. The cells somehow pass through peripheral tolerance and cause a self reaction. Autoimmunity is determined by genetics, environmental, and hormonal effects that contribute to the disease. Many autoimmune diseases have been studied and examined but the biology behind them are not completely understood.

A major pathway is the CD40-CD40 ligand (CD154) because it is needed to activate many adaptive immune cells such as DC's, B cells, and T cells. This interaction has multiple functions and as costimulatory molecules, they are upregulated in several autoimmune diseases for example SLE (systemic lupus ertyhematosus). It was thought that disabling this interaction could be a new avenue of therapy for autoimmunity.

Caption: The image to the left shows the basic interactions between immune cells in the body when trying to get rid of an antigen. The same interactions occur in autoimmune diseases where the body is attacking self antigens. In this picture, we can see the CD40-CD40L (CD154) interaction described above between a dendritic cell presents antigens and a CD4+ T cell which aims to help the immune response by releasing cytokines.

CD40 provides a help signal to dendritic cells to maturation. The duration of the signal assists in determining the function of the dendritic cells which is displayed in this study. There are various other proteins that can trigger dendritic cells to become activated and thus spark an autoimmune response such as heat shock proteins. These can skew a response using cytokines, interleukins, and different factors. In a mouse model, diabetes was induced when CD40 expression on bone marrow was necessary for heat shock protein induced activation. 

This reaction of CD40-CD154 is not only found in dendritic cells but it also impacts T cells by priming them, polarizing T cells to a certain response.

Caption: The basic polarization of T cells is shown in the following image with different cytokines determining the Th cell developed. 

Th (T helper) cells only express CD154 after they have been stimulated by an antigen which allow them to have this interaction of CD40-CD154; unfortunately over expression of CD154 can lead to autoimmune diseases. This not only occurs in T cells but in B cells as well. The CD40-CD154 interaction enhances CD86 expression on B cells in autoimmunity specifically SLE disease which is a hypersensitive reaction. CD86 contributes to the presentation of self-antigens to T cells thus inducing a negative immune response by the body. 

Ring The Alarm! Granulysin’s Role in Recruiting Dendritic Cells

Our body puts up a tough fight against a constant barrage of invaders. Our first line of defense against invading pathogens is referred to as our “innate defense” and consists of a rapid response by NK, NKT and ϒδ T cells (1). These cells respond much quicker than T and B cells which are activated later if the infection persists. However, this secondary “adaptive response” requires the recruitment of dendritic cells (DCs). DCs are able to activate T and B cells highly specific for that antigen to order to mount a more focused response (1).

A recent study by Tewary et al. 2011 looks specifically at the role NK cells plays in the secondary adaptive immune response, specifically via the release of granulysin.

The GNLY gene produces the antimicrobial protein granulysin, which is initially 15 kDa in size. NK cells secrete this initial form that can be further cleaved into a smaller form, 9-kDa. The 9-kDa form is stored in the granular compartment of NK cells. The contents of the granular compartment contain toxins that cause cell death when released (degranulation) onto their pathogen targets (1) as part of the innate response.

However, a previous study also showed that GNLY participates in the adaptive response by recruiting dendritic cells (DCs), NK cells, and T cells (2), though the mechanism by which it was able to do so was unclear. Molecules that exhibit this recruitment capacity are referred to as alarmins.

In Tewary et al. 2011, the mechanism by which GNLY is able to recruit DCs and T cells and promote an immune response is elucidated.

Finally Reaching Out: Ralph Steinman Wins Nobel Prize for Fingery Find

This past Monday the Nobel Prize in Physiology or Medicine was awarded to a very deserving candidate: Ralph Steinman. Steinman, however, never received the final good news, as he passed away from pancreatic cancer just three days prior to the announcement. He is the first laureate to be awarded a posthumous Nobel Prize, which is against the regulations of the foundation.¹ Steinman was awarded the prize for his discovery of dendritic cells, “a kind of sentinel”² looking for pathogens in the body.

Dendritic cells (DCs) were given their descriptive name due to their physiological appearance, with many finger-like extensions that protrude from the main cell body increasing the surface area of the cell for the intake and output of information from and to other cells in the body. Dendritic cells come in several different types. Some DCs, called “migratory” DCs, roam the periphery of the body searching for foreign substances or dead cells that they can engulf. The internalized substances are then processed in the DC and presented on the cell’s surface, so that a passing B or T cell might recognize the presented pathogen and go find it in the body to fight. “Resident” DCs reside in the lymph nodes of the body, sampling the lymph for pathogens and undergoing the same pathways of processing and presenting pathogens as the migratory DCs.

Steinman first discovered dendritic cells in 1973, and died three days before his work was recognized with the highest of honors: the Nobel Prize. Upon his initial discovery, the scientific community was hesitant to accept Steinman’s finding, as his results did not seem reproducible. Reproducibility is the hallmark of a significant scientific finding, and so it was not until many years later, when dendritic cells were isolated in large quantities, that Steinman’s finding was widely accepted and celebrated. Modernly, dendritic cells are arguably considered to be one of the most significant elements of the immune system. Dendritic cells are antigen-presenting cells (APCs), which gives them the capability to express both endogenous and exogenous antigens, and they also possess the ability to activate B and T cells, which have cytotoxic capabilities.

Something to squeak about: “Dirty” Wild Mice may be Better Models for Immunological Studies

When we become infected with a virus or develop cancerous cells, our bodily friend group expands as natural killer cells (NK) come to our defense to protect us from these nasty invaders. Like a trained body guard, the NK cells immediately respond to enhance our immune system by defending us against microbes and tumors in our bodies. They have granules in their cytoplasm which host proteins such as perforin and proteases (granzymes) which are released near a pathogen. Upon release, the perforin perforates holes in the cell membrane of the pathogen through which the granzymes and associated molecules can enter to induce apoptosis[o1] .  Interferons and macrophage-derived cytokines activate NK cells to contain viral infections while the adaptive immune response is generating antigen-specific cytotoxic T cells.

Much of what we have learned about immunological processes such as these have stemmed from research on animal models. Mice serve as an important model organism to study molecular mechanisms in immunology.  NK cells from laboratory mice respond poorly to stimuli unless the mice are treated with toll-like receptor (TLR) agonists, cytokines or infection. Previous studies indicate that NK cells are extremely low in lymph nodes (LNs), to lack perforin or granzyme B (Gzmb) and to die prematurely in cytokine absent cultures (Fehniger et al. 2007; Long 2007). This starkly contrasts with NK presence in humans which are profuse in LNs, contain perforin and Gzmb and survive better. Previously these discrepancies were attributed to the fact that the differing species had varying NK cells.  NK cells were believed to be short-lived and innate.  Recent studies, however, suggest that NK cells may be live longer than was previously thought.

Dendritic Cells Directly Engage Cancer

As members of the Colgate community know, especially in light of the recent passing of student Vic Krivitski ’12, cancer affects everyone’s life. Less apparent are the specific ways in which the immune system acts to mitigate cancer. To elucidate these mechanisms, a recent study in the Journal of Immunology investigates the role dendritic cells (DCs) play in killing cancer cells.

Cancer is a condition of uncontrolled cell growth. Not only are cancer cells good at promoting their own proliferation, but also they are proficient at avoiding the immune system. DCs are immune cells that have two characteristic functions, both of which can be thwarted in the presence of cancer (1). Typically, DCs act as antigen presenting cells (APCs), presenting antigens, pieces of pathogens, to lymphocytes to activate them. They can also secrete cytokines, signal molecules, that tell other cells to combat pathogens. Less well understood is DCs’ ability to act as killer DCs (KDCs), by directly engaging pathogens, and how this function is affected by cancer.

Larmonier and Bonnotte et. al. attempt to enhance the understanding of human-derived KDCs (hKDCs). KDCs have been examined previously in rats, mice, and humans (2,3,4) but this investigation provides finer details of hKDCs’ effective activation, mechanism for inducing cancer cell death, and ability to retain characteristic DC function.

hKDCs were prepared by exposing monocyte-derived DCs from cancer-free and stage IV cancer participants to low concentrations of an activating molecule, LPS. These hKDCs were found capable of killing several tumor lines (including HeLa) and reducing cancer cell viability up to 80%. This is a particularly exciting result because DCs derived from cancerous environments were found to be as effective as those from cancer free environments.

The mechanism for cancer cell death was elucidated via several experiments. ESR spectroscopy, which measures free radicals, showed that hKDCs produce reactive oxygen species (ROS). Decreased anti-tumor activity in the presence of FeTPPS, a free radical inhibitor, confirmed this result. When cancer cells and hKDCs were separated by a semi-permeable membrane, decreased anti-tumor behavior was observed suggesting that hKDCs kill cancer cells via direct cell-cell contact. Additionally, a staining experiment that allowed visualization of the dying cells as well as the death of certain apoptosis-resistant cancer cells suggests that the cells die by necrosis (catastrophic cell death) rather than apoptosis (programmed cell death). This, too, is promising because cancer cells are particularly adept at avoiding apoptosis, so a treatment that includes a necrotic mechanism might be effective against these cells.

Furthermore, the authors demonstrate that hKDCs retain their original DC functions: they are still able to present antigens (even those from cancer cells they kill themselves!) and produce cytokines. These promising results are not problem free and will require further study. hKDCs’ ability to kill certain non-cancerous cells requires an extensive investigation of the interactions between hKDCs and healthy cells. (It is important to note, however, that T lymphocytes were not killed by hKDCs. In fact, hKDCs produce cytokines that help T lymphocytes resist death.) Similarly, the mechanism of cell death itself is a danger to healthy cells, because necrosis releases dangerous toxins. Nevertheless, this study makes strides in understanding the relevancy of hDKCs for potential cancer treatments and may aid in the development of such treatments.

Reference: Lakomy, D., Janikashvili, N., Fraszczak, J., Trad, M., Audia, S., Samson, M., Ciudad, M., Vinit, J., Vergely, C., Caillot, D., et al. 2011. Cytotoxic dendritic cells generated from cancer patients. J. Immunol. 187(5): 2775-82.

Other Citations:

(1) Brown, M.P., Diener, K.R., Fraser, C.K., and Hayball, J.D. 2010. Unraveling the complexity of cancer immune system interplay. Expert Rev. Anticancer Ther. 10: 917.

(2) Fraszczak, J., Trad, M., Janikashvili, N., Cathelin, D., Lakomy, D., Granci, V., Morizot, A., Audia, S., Micheau, O., Lagrost, L., et al. 2010. Peroxynitrite-dependent killing of cancer cells and presentation of released tumor antigens by activated dendritic cells. J. Immunol. 184: 1876–1884.

(3) Chauvin, C., Philippeau, J. M., He ́mont, C., Hubert, F. X., Wittrant, Y., Lamoureux, F., Trinite, B., Heymann, D., Re ́dini, F., and Josien, R. 2008. Killer dendritic cells link innate and adaptive immunity against established osteosarcoma in rats. Cancer Res. 68: 9433–9440.

(4) Hill, K. S., Errington, F., Steele, L. P., Merrick, A., Morgan, R., Selby, P. J., Georgopoulos, N. T., O’Donnell, D. M., and Melcher, A.A. 2008. OK432- activated human dendritic cells kill tumor cells via CD40/CD40 ligand interactions. J. Immunol. 181: 3108–3115.