Thursday, June 7, 2012

CD95/CD95L Application to Tissue Grafts

      My cancer presentation delved into discovering the role of CD95/CD95L in tumor counterattack and the validity of such an escape mechanism. While researching this mechanism I came across an older paper that suggested future studies in this area may be able to apply CD95/CD95L to increasing the stressfulness of tissue grafts. So I got onto google and searched to see if this mechanism had been applied to tissue grafts yet, and sure enough there is an ongoing effort to utilize cancer's trickery to aid the medical community. Here are the results of my research, but first, some background information on CD95/CD95L, allogeneic immune response and the current therapies used to treat side effects of tissue grafts.

       CD95/CD95L is a ligand and receptor set typically used by T-cells to induce apoptosis in pathogens or non-self cells. Here is a simplified version of how this system works.

      First the T-cell receptor (TCR) recognizes the major histocompatability complex (MHC) on the cell (in this case a tumor cell) and engages the CD95L to the CD95 receptors on the tumor cell. This induces a signal cascade that results in apoptosis of the cell (tumor). For more details on this process visit our wiki.
      An allogeneic immune response occurs when dendritic cells (DCs) from both the host and donor present MHC molecules. Typically this immune response comes in the form of host T-cells searching and then destroying (inducing apoptosis) of the non-self cells (there is also and element of donor T-cells seeking host cells to destroy.) The common name for this response is graft vs host disease or GVHD.
     Until now several strategies have been developed to prevent or treat GVHD. The first is the ex vivo removal of donor T-cells prior to infusion. Three other methods have been used to modify T-cells by triggering clonal anergy of effector T-cells, controlling the expansion of T-cells and blocking T-cells adhesion molecules. The goal of this experiment is to selectively kill alloreactive T-cells.

New Therapy:
      This new study is landmark because it is trying to take the guess work and generality out of current therapies. The idea of this experiment is to develop a sort of 'buffer' that would work between host and donor tissues to prevent both host and donor immune response by selectively inducing apoptosis in alloreactive T-cells.
       To create the buffer DC cells from mice were taken and exposed to the cDNA of CD95L.
When the 'killer' DCs were implemented between donor and host cells of a syngeneic nature (nearly compatible tissues) the DCs were capable of suppressing the immune response. However, when the same buffer was implemented between tissues of allogeneic nature, only about 70% of T-cells were destroyed. This led to researches theorizing that a hybrid of donor and host DCs were necessary to suppress the immune response in allogeneic conditions. Through a complex mechanism (see the paper for details on the method), researches were able to develop the killer hybrid DCs and continue testing into allogeneic conditions.

Positive Results In Vivo:
       The first test was to see how well killer hybrid DCs could inhibit the immune response compared to control hybrid DCs (DCs without the CD95L cDNA mix). The results from the study are seen in the figure below.
      Here BALB/c (recipient) and A/J (donor) are two allogeneic mice types. The '1st donor' is an injection to sensitize the immune system to the non-self cells. PBS is just a saline solution. We see that if the recipient is sensitized with A/J, and then treated with saline and control DCs, there were similar levels of swelling. However, mice sensitized and then treated with killer DCs had similar levels of swelling to those mice that were not sensitized first. The immune response is not as strong with unsensitized mice, so for the level of swelling to be similar in this case shows that the killer DCs have a measurable effect on the immune response.

      The next test run in vivo measured what sort of benefit killer hybrid DCs lent to staving off the symptoms of GVHD.  3 million spleen cells from allogeneic donors were grafted onto the spleen of host mice and then injected with either saline (open circles) or killer hybrid DCs (closed circles) on days 0, 3, 5, and 7. A Kaplan-Meier plot is seen below illustrating the results.
     Clearly, despite both sets of mice dying in the end, the mice treated with killer hybrid DCs did markedly better, keeping the mice alive an additional 12 days. 

Negative Results In Vivo:
       Despite the positive results seen in the tests above, there still appears to be some pretty severe limitations on this therapy. In the test below the recipient was first sensitized and then on days 7, 14 and 60 given tissue grafts of allogeneic cells. They were either treated with killer hybrid DCs or saline.

      The results show that mice sensitized with A/J and then treated with killer hybrid DCs at first showed less swelling, but by the 3rd challenge and day 60 the swelling had increased to match the mice treated with just saline. This test illustrates that periodical injections of killer hybrid DCs are needed to maintain the benefits (the mice were injected with treatment on days −6, −4, 0, 3, and 6). It is even possible that despite constant injections, the immune system may adapt to the therapy and prove killer hybrids ineffective.

       A second limitation of this therapy is the size of the tissue graft that can be adequately treated. Below is a Kaplan-Meier plot showing a similar test to 'A,' but this time the size of tissue sample is changed in each graph.
      Notice that as the number of cells/animal increase, the difference between the two treatments decreases. By the time 100 million cells are grafted onto the spleen on the mice, there is almost no difference in additional days of survival. Clearly there is a limit to what size of graft can be aided by this current treatment.

       In conclusion, research so far has yielded beneficial results; however, the current level of understanding is not enough to guarantee this treatment a place in future therapies. This treatment has proven to 'wear off' before 60 days in addition to being unable to treat a large (>30 million cells/animal) tissue graft. Perhaps with further testing and development this mechanism will prove to be useful in a therapeutic sense. I conclude that the real benefit of this study was to provide frameworks for clinical applications of the killer hybrid DCs technology in addition to furthering understanding of CD95/CD95L pathways. Hopefully with further research into this treatment, further understanding of tumor counterattack will emerge.  Possibly by studying how the immune system is capable of adapting to killer hybrid DCs treatment, researchers can apply this to boost the immune system to stop tumor counterattack. 


Strand, S. (1998). Immune evasion by tumors: involvement of the cd95 system and its clinical implications.  Molecular Medicine Today.