Matt Boyle, Entry #4, Do We Only Cure Dogs? "Nayyyyy."

After all this talk about CD40 and K562s that are irradiated and culturing the cells, I realized I have never explained to you guys what CD-40 B Cell activation is.  My bad.   So here we go.

Cd40 B Cell activation is a method by which B Cells become antigen presenting cells.  The binding of CD40 to CD40 Ligand activates the cells, thereby increasing the ability of the cells to present foreign antigen to the immune system.  In the, the PBMCs of an organism are extracted and cultured with these feeder cells (from the K562 cell line) expressing CD40L.  In the presence of the cytokine interleukin 4 (IL-4) the activated cells proliferate, allowing for a large number of cells to be grown from a small number extracted from a blood sample.  This is extremely advantageous because, as each vaccine is specific to the host the blood was taken from, just a small amount of B cells from a small blood sample can grow into a huge amount of antigen presenting B cells that will increase the immune-response to the cancer.

The problem is (and the efforts of my project are based around) the feeder cells we have in the lab express HUMAN CD40L.  Canine CD40 recognized the human CD40L but we do not know if the horse CD40 will.  I performed a blast search on the homology of canine and equine CD40 and the proteins are approximately 70% similar so nothing is guaranteed.  Should the CD40 of the equine cells bind with the human CD40L, the cells will be activated and their proliferation will be sustained in the lab until B-Cell count reaches a threshold of viable cells able to be infused back into the horse.  Then the cells will be electroporated with the RNA from a tumor sample of the horse removed via a biopsy.  The B Cells will then present the tumor RNA as an foreign antigen to the immune system, and upon re-infusion into the horse (the same horse that the B Cells came from) its immune system will recognize the tumor as foreign, thereby educating it to recognize and kill the rest of the tumor cells.

An animation of the CD40 B Cell Activation. The little orange things on the sides is RNA being electroportated into the cells before re infusion of the B Cells into the patient 

The science behind the protocol is truly fascinating but that is only half of the reason I am enjoying EXP so much. The other half is because of the people I get to work with.  Dr. Mason Is truly hilarious; for example, when the horse blood came in she was walking around saying “NAYYY.”  Dr. Mason also just got a full-time vet to help her with the trials in the clinic (her name is Martha and she just graduated from Cornell’s Vet School).  We all went out to dinner in Philly for Martha’s first day where Dr. Mason was telling us about how she was invited to Richard Lichter’s dog’s birthday party on Saturday.  Richard Lichter is a multi-millionaire who owns Mel Gibson’s old estate in Greenwich Connecticut with 16.5 bathrooms) and his goal in life is to cure equine lymphoma.  He is an enormous benefactor for Penn’s veterinary research and has a party date set (April 16) for the celebration of Dr. Mason having cured canine lymphoma.  Upon hearing this, Dr. Mason decided we must get to work as cancer is certainly not cured yet. We sat outside at dinner and Dr. Mason was directing a girl to parallel park next to our table when a group of people came out of the restaurant in a huge fight.  People were screaming at each other and Dr. Mason did not miss a beat as she stood there like one of those people on the runways at airports helping this girl parallel park.  It was probably the funniest moment from my time in the lab.

As for the horse cells, they look horrendous. But not really.   If you look through the microscope at the K562 Wells in relation to the KtCD40L Wells, there is significant more clumping in the CD40L wells, leading one to believe the B Cells were indeed interacting with the human CD40 protein.  However, upon analysis, the B Cell populations were almost completely dead (as you can see in the flow graph).  Despite this failure, the way the cells looked through the microscope was very promising.  The cells seem to be interacting with the protein but not proliferating, which could be very closely related to an insufficient amount of the cytokine IL-4 that causes proliferation or perhaps there is insufficient serum, insulin, or other component of the growth media.

The K562 (left) and KtCD40L (right) wells as they appeared through the microscope.  There is obvious clumping going on in the KtCD40L wells; significantly more than in the K562 wells, leading one to believe that the equine CD40 is indeed interacting with the huamn CD40L.

 These the flowcytometry graphs pertaining to B Cell population in one horse (R600). You can see from the day the bloods arrived (left) that the B Cells made up 17% of the living PBMCs, but on day 7 when the cells were flowed again, the percentage of B Cells had shrunk to only 3.12% of the population.  This is an opposite result to what we thought was the case by looking at the cells through the microscope.

The plan from here is to get new horse blood and determine which components we will change from the media or in what is added to the wells to initiate B-Cell proliferation.  Now I am staying for an extra week to have some time to see round two through to completion.