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Sunday, December 2, 2018

Using Induced Regulatory T cells to prevent Graft vs Host Disease

Based on Stabilization of Foxp3 by Targeting JAK2 Enhances Efficacy of CD8 Induced Regulatory T Cells in the Prevention of Graft-versus-Host Disease 
    Written by Supinya Iamsawat working in Dr. Xue-Zhong Yu’s lab within the Department of Microbiology and Immunology at the Medical University of South Carolina. Dr. Xue-Zhong’s research profile can be found here  

            Transplanting healthy donor tissue into sick patients is becoming more and more prevalent as a treatment option. This treatment works for many different afflictions, from organ failure, to burn victims needing skin transplants. Even leukemia can be treated with transplantation.1 Leukemia is a name for cancer in blood cells, in which blood cells replicate abnormally. The cancerous blood cells cannot function normally, and they hinder the production of normal blood cells creating a multitude of symptoms. For example, a limited ability to fight off infection since leukemia interferes with white blood cells that are responsible for fighting off infection. More on Leukemia and its symptoms can be found here. 2
            To treat leukemia, patients can receive a stem cell transplant from a donor meant to help restore healthy blood cells. This means taking the donor’s healthy stem cells and placing them inside the patient. Once the donor stem cells are inside the patient’s body they will turn into blood cells, like red blood cells that carry oxygen and white blood cells that fight infection. The process of introducing donor stem cells into a patient’s body is called hematopoietic stem cell transplantation.3 Some of the donor T cells become functional white blood cells that fight and kill the cancerous blood cells of the patient. This is called the graft vs leukemia effect, in which the donor stem cells are called the “graft” and the “host” refers to the sick patient. However, the benefits of this are limited by the side effect in which the T cells resulting from the stem cell transplant start attacking the patient’s normal cells in a condition called graft vs host disease.4 This disease can cause rashes and liver damage, with varying severity. More on graft vs host disease can be found here.5  Current research is aimed at figuring out ways to maximize the graft vs leukemia effect of the stem cell transplantation, resulting from the new T cells killing the cancerous cells, and minimize the graft vs host disease where the new T cells harm the patient.
            In this paper Iamsawat and colleagues show that treatment with an alteration of an induced regulatory T cell can relieve graft vs host disease, while maintaining the graft vs leukemia effect in mice. Regulatory T cells6 have been used to alleviate graft vs host side effects in mice previously.7 The function of regulatory T cells is to prevent healthy cells from being harmed during the process of fighting against the foreign material, through controlling and limiting the immune response. Induced regulatory T cells are cells that first exist as regular, infection fighting T cells, before they become regulatory. Iamsawat and colleagues used the induced regulatory T cell that comes from a CD8 T cell,8 hence the name, CD8 induced regulatory T cell (CD8 iTreg). These has been shown to have significant graft vs leukemia effects in mice, however, CD8 iTregs struggle to treat the graph vs host side effects because they often lose their expression of Foxp3 during immune responses. Foxp3 is a gene, responsible for a large part of the induced regulatory T cells’ suppressive function. CD8 iTregs are prone to losing the expression of Foxp3 in the presence of inflammation signals which are chemicals secreted by immune cells, used to recruit more cells to assist in fighting infection.8 This creates a problem because when the CD8 iTregs arrive at the immune response, instead of suppressing it as intended, they lose their expression of Foxp3 and in doing so, they can turn into T cells that contribute to the excessive immune response. Iamsawat and colleagues have engineered CD8 iTregs that lack a gene called JAK2 which is involved in the pathway of losing Foxp3 expression and turning into an effector cell.9 They hypothesized their JAK2-/- CD8 iTregs will alleviate graft vs host side effects, while maintaining the beneficial graft vs leukemia effects because these engineered T cells will not lose expression of Foxp3, and thus will maintain their suppressive function throughout the immune response preventing the harm of healthy cells. 
            The researchers first examined whether their engineered JAK2-/- CD8 iTregs expressed more Foxp3 than normal CD8 iTregs using flow cytometry. Flow cytometry is a method of detecting proteins that are expressed on the surface of the cell. An in depth explanation of flow cytometry can be found here.10 They found that their JAK2-/- CD8 iTregs expressed significantly more Foxp3 than the normal CD8 induced regulatory T cells. Next, they tested the ability of the CD8 to alleviate graft vs host disease in mice. Mice were radiated to decrease their amount of healthy blood cells, mimicking the condition that humans are in when they receive donor stem cells. They then were injected with healthy mice T cells to induce graft vs host disease. The mice that were treated with the researcher’s JAK2-/- CD8 iTregs survived at a much higher rate (about 70% of the mice survived beyond 80 days), than mice that were treated with normal CD8 iTregs, in which all of the mice died within 60 days. Further, their data suggests that the JAK2-/- CD8 iTregs are successful in treating graft vs host disease because JAK2-/- CD8 iTregs influence the transferred T cells to start expressing Foxp3 themselves and become regulatory T cells, contributing to the prevention of immune responses directed at the host’s healthy cells. 
            After showing that their JAK2-/- CD8 iTreg cells do indeed treat graft vs host disease, they needed to show that the cells maintained their effectiveness at killing cancerous cells. The researchers injected the mice with leukemic (cancerous blood) cells on the same day that they injected the various types of T cells. The specific type of leukemic cell that they used expresses a protein allowing the researchers to use flow cytometry to monitor the growth of these cancerous cells over time. The group of mice that were not given any T cells at all, showed tumor growth at 30 days after they were given the cancerous cells, ensuring that the leukemia cells create tumors in mice if not treated with regulatory T cells. The group that was given normal CD8 iTregs did not show signs of tumor growth, however these mice suffered from graft vs host disease that eventually killed all of the mice around the 40th day after receiving the cancer cells. The group of mice given JAK2-/- CD8 iTreg cells maintained the graft vs leukemia effect of regular CD8 iTregs, as there were no signs of tumor relapse, but also significantly alleviated some of the graft vs host disease because about 30% of the mice survived past the 80th day after receiving cancer. With this experiment, the researchers show that removing the gene JAK2 from the CD8 iTreg cell helps to alleviate graft vs host disease, while maintaining the ability of normal CD8 iTregs to fight off cancerous cells. 
            The researchers did one last experiment mirroring the last one, but this time used a drug called pacritinib that is an inhibitor of JAK2. Inhibiting JAK2 in this way, showed similar results as treatment with JAK2-/- CD8 iTreg cells. Mice treated with CD8 iTregs and pacritinib together showed decreased graft vs host disease and maintained the graft vs leukemia effects. This paper suggests that inhibiting JAK2 is a plausible method for treating graft vs host disease in humans, however more research must be done aimed at revealing, with greater certainty, the effectiveness that this would have in human patients. In their mice model of the graft vs leukemia effect of JAK2-/- CD8 iTregs, about 30% of the mice survived being given cancerous cells with no signs of tumor growth. Though this is significantly higher the 0% survival rate for the other groups, it is not a high percentage in itself. Furthermore, there are four main types of leukemia and various subtypes, so more research must be done to see if the effects of JAK2 inhibition are present throughout all types. 
            Iamsawat’s work provides a step forward towards the goal of separating the damaging graft vs host disease from the beneficial graft vs leukemia effect. The effect of inhibiting JAK2 can be kept in researcher’s minds as further research goes into enhancing the ability of transplanted donor stem cells to combat cancer, while avoiding healthy tissue. The more efficient transplanting stem cells becomes, the more it can be utilized to assist in donor tissue transplantation, and to keep cancers in remission. Remember, you can check out this paper and other research by Dr. Xue-Zhong’s lab yourself using the links at the top!

References
1.     Dickinson, A. M., Norden, J., Li, S., Hromadnikova, I., Schmid, C., Schmetzer, H., &
Jochem- Kolb, H. 2017. Graft-versus-Leukemia Effect Following Hematopoietic Stem Cell Transplantation for Leukemia. Frontiers in immunology 8: 496. Retrieved from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461268/ 
2.      “Leukemia.” 2018. Retrieved from:     
3.      Franks, I., Biggers, A. 2017 June 26. “What is hematopoietic stem cell transplantation?” 
4.      Kolb, H. J. 2008. Graft-versus-leukemia effects of transplantation and donor 
lymphocytes. Blood, 112: 4371-4383 
5.      “About graft versus host disease (GvHD)” 2017. Retrieved from: 
6.      Corthay, A. 2009. How do regulatory T cells work? Scandinavian journal of
immunology, 70(4): 326-36. Retrieved from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2784904/ 
7.      Zhang, N., & Bevan, M. J. (2011). CD8(+) T cells: foot soldiers of the immune
system. Immunity, 35(2): 161-8. Retrieved from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3303224/
8.      Beres, A., R. Komorowski, M. Mihara, W. R. Drobysk. 2011. Instability of Foxp3 
expression limits the ability of induced regulatory T cells to mitigate graft versus host disease. Clin. Cancer Res. 17: 3969–3983
9.      Betts, B.C.D. Bastianet al. 2018. Targeting JAK2 reduces GVHD and xenograft 
rejection through regulation of T cell differentiation. Proc. Natl. Acad. Sci. USA 115: 1582–1587.
10.   Jahan-Tigh, R.R., C. Ryan, et al. 2012. Research Techniques Made Simple: Flow 
Cytometry. Journal of Investigative Dermatology. 132(10): 1-6. Retrieved from: https://www.sciencedirect.com/science/article/pii/S0022202X1535487

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