Development of a model for adult T cell leukemia in HTLV-1 infected humanized mice
Faculty Mentor: Dr. Brad Berges, Department of Microbiology and Molecular Biology
Millions of people in the world are infected with human T-lymphotropic virus type-1 (HTLV-1). As the name states, this virus attacks the T cells of the infected organism. Though many of these individuals will remain asymptomatic throughout life, a small percentage will develop adult T cell leukemia/lymphoma (ATLL), a rare and understudied form of cancer. The latency period, or amount of time between infection and detection of cancer, is generally 20-40 years before infected patients begin developing cancer.
HTLV-1 infects T cells. There is no good animal model in which HTLV-1 and ATLL can be effectively studied. The latency period length is the reason that using the humanized mouse model is crucial in being able to identify primary target cells that are infected by HTLV-1 and determining factors that promote virally induced transformations. HTLV-1 cannot infect normal mice due to the lack of a viral receptor on mouse cells. Humanized mice are transplanted with human immune cells and thus can likely be infected with HTLV-1. One previous study was conducted in this area, but the paper was retracted due to data manipulation. That study showed a latency period of HTLV-1 in humanized mice of approximately 18-20 weeks which is much more manageable. Using a humanized mouse model also offers insight into how HTLV-1 interacts with a human-like immune system which cannot be achieved using other methods because key immune cells from the mouse have been knocked out and replaced with human equivalents, including T cells which are infected by HTLV-1.
Methodology and Results
Many different techniques were employed to examine the HTLV-1 infection itself as well as the development of cancer. We first prepared the virus by growing it up in MT-2 cells. This cell is able to be chronically infected by HTLV-1 and therefore allows the production of many viral copies. Next, we quantized the number of viral copies per milliliter through a multistep process. This process involved removing the genome from the virus, converting it to DNA from RNA, replicating a very specific portion of the genome, placing it in a plasmid with a known sequence, growing the plasmid in E. Coli, sequencing the plasmid to ensure that the correct part of the genome was retrieved, and finally, using quantitative PCR (qPCR) to determine the concentration of our virus stock.
Once the stock concentration was known, we were able to infect the mice with confidence that enough virus was being injected to take hold and maintain an infection. 15 mice were split into 3 groups of 5. The first group consisted of non-humanized mice that were challenged with HTLV-1. The second group was made of humanized mice that received an injection of buffer solution, not HTLV-1. The third group consisted of humanized mice that were challenged with HTLV-1. The first two groups were controls and the third group was the experimental group.
After infection, the mice were bled weekly. On the odd weeks post infection, qPCR was performed. Blood samples were separated via centrifugation. RNA was extracted from the plasma. This was done because any viral genome found in the plasma would be free floating and in the original RNA form. DNA was extracted from the blood pellet as this pellet would include a number of cells that could have been infected by HTLV-1 and therefore, the virus would have been going through the replication process which involves conversion of its RNA genome to DNA. The DNA and RNA would then be prepared for qPCR and the results analyzed.
Figure 1 shows the results that we received from qPCR. As can be seen from the figure, RNA was mostly found for on-humanized mice in the later weeks after infection. This was an interesting result. One possible reason for the non-humanized mice to be able to sustain an infection is that they are heavily immune deficient and therefore the virus may have been able to survive simply because there was nothing in the mice that could attack it effectively. Looking at the DNA results, we see that the majority of the positive readings are the humanized infected mice, especially towards week 11 and week 13. This confirms that HTLV-1 was able to replicate and maintain an infection in the mice. The importance of this is that we now know that the strain of mouse that was used, a Rag2-/-γc-/- mouse, is able to be infected by HTLV-1, a human virus.
On the even weeks, flow cytometry was performed. This is a process that marks specific cells and allows the researcher to compare the presence of different cells in a specific circumstance. We knew that HTLV-1 caused an over proliferation of CD4+ T cells. Expecting the amount of CD8+ T cells to remain the same, we determined to compare the percentage of each of these cells to find out if the CD4+ T cells were expanding more than is normal. Figure 2 shows the results. Indeed, we see an upward trend in the amount of CD4+ T cells compared to CD8+ T cells in the humanized infected mice. It is important to note that there were no significant readings from the non-humanized infected mice, showing that there is no increase in CD4+ T cells. This is because the non-humanized mice have no human immune cells so nothing was marked in the flow cytometry process. It is also interesting to note that the humanized uninfected mice also increased in CD4+ T cells similar in fashion to that of the humanized infected mice.
Results and Conclusion
In conclusion, we have seen from the use of qPCR and flow cytometry that HTLV-1 is able to infect and maintain itself in a Rag2-/-γc-/- mouse. We have also shown trends that the virus acts similar in this strain of mice to that of humans by causing the CD4+ T cells to proliferate more than usual.