SDSC meeting Mar. 18, 2015

This was the first SDSC meeting of the year over at the Green Acre Eatery on the Eli Lilly campus. Carina Torres started off the meeting by talking about the upcoming SoCal flow cytometry meeting in Irvine Apr. 22-24. It will be a good chance to hear the latest research in flow cytometry as well as an opportunity to take some classes in flow cytometry being offered by FloCyte and ExCyte. The FloCyte class will be on Compensation and Multiparameter Strategies and the ExCyte class will be on Advanced Data Analysis. Instead of introducing new members to the group, Carina introduced Cheryl’s new baby who is just over a month old.

Mike Stadnisky from FloJo gave a talk on data analysis, integrating high content data into FloJo. He showed some comparison data of automated analysis versus manual gating. Using machine learning, they are taking the subjective gating out of analysis using an automated algorithm with snap gating. They have now added a program called 4th wall which organizes the data with the reports making the workflow much smoother.

Gerhard Wingender from LJI gave a talk on setting up a 17 color T cell panel. His project is looking at invariant natural killer cells from frozen patient PBMCs. In deciding on his panels, he needed to make up a wish list of antigens and decide on which ones would work on frozen PBMCs, as well as color selection based on spectral overlap. Websites such as BD’s panel designer and Biolegend’s panel selector help to select appropriate dyes. Using all of these tools and considerations, each antibody is individually titrated before adding into the full panel.

The next SDSC meeting will be held in May. The date will be announced as it gets closer. We look forward to seeing you there.

SDSC meeting July 23,2015

There was a great turnout of people at last Wednesday’s BioLegend-sponsored SDSC meeting, which took place at the beautiful Green Acre Eatery within the Lilly Biotechnology Center in La Jolla, CA. Following some friendly networking and intellectual merriment, attendees were treated to a flow content rich presentation by Dr. Michael Betts (Department of Microbiology, Perelman School of Medicine, University of Pennsylvania) regarding his work on CD8+ T cell function during HIV infection.
Carina Torres commenced by introducing the new people she met at the happy hour preceding Dr. Betts’ presentation. Since the flow cytometry community in San Diego is a very small and tight-knit group, she plans to continue this ritual at each meeting. John Nolan (La Jolla Bioengineering Institute) then informed us of the upcoming Systems for Cytometry in Regenerative and Personalized Medicine Symposium, a three day local meeting this October. The symposium normally takes place at the Asilomar Conference Center in northern California, but will be at the La Jolla Shores Hotel in San Diego this year. There will be a day of tutorials and the symposium will begin on Oct. 30.
The reason we are having 2 meetings in a row this summer is because Dr. Betts was in town for a conference. Dr. Betts studies the role of CD8+ T cells in HIV infection and the title of his presentation was “Flow cytometric assessment of CD8+ T cell function during acute and chronic HIV infection: implications for vaccines and cure based strategies.”
Dr. Betts began by describing the normal role of CD8+ T cells in infection. It is critical for the cytotoxic lymphocytes (CTLs) to clear out the reservoirs of HIV in the lymph nodes. He shows that the cytotoxic activity of CD8+ T cells is regulated by perforin through delivery of granzyme B. Since perforin is required for granzyme B transfer, he found that it was only important to monitor perforin. He setup panels of antibodies to specific lineage cell types, memory markers, transcription factors, and functional markers to monitor changes upon HIV infection. Using samples from a U.S. Army cohort of high risk individuals, Dr. Betts’ team stimulated the cells with peptide pools and monitored patients before and during different stages of HIV infection. They monitored perforin vs. HLA DR expression pre and post infection at different stages and were able to see a massive expansion of perforin+/CD8+ HIV specific T cells, which quickly reverted back to a pre-infection state around 28 days post initial infection. Moreover, they did not observe these changes in B cells or CD4+ T cells, and neither yellow fever vaccination nor influenza infection induced a similar response. Along with detection of Gag, a marker for HIV, these results provided additional evidence that the change in the perforin+/CD8+ T cell population was HIV specific. They also chose to monitor T-bet and Eomesodermin, transcription factors critical for CD8+ T cells to become effector cells and thus obtain cytotoxic potential. Dr. Betts also explained how the particular conjugate of their T-bet antibody was important to visually distinguish T-bethigh (found to be effector CD8+ T cells expressing perforin) from T-betlow (found to be central memory cells expressing granzyme B but not perforin) populations. They found that the population of perforin+ CD8+ T cells decreased the amount of T-bet with prolonged infection.
In the second part of his talk, Dr. Betts discussed T cell function in the lymph node (cervical). He first addressed T follicular B helper (TFH) cells, which are CD4+ T cells and are massively expanded upon HIV infection. He then described his findings from an LCM study designed to address whether CD8+ T cells in the lymph node are cytolytic. It turned out that CD8+ T cells from the spleen and peripheral blood were cytolytic, but those from the lymph node were not. Interestingly, he found that CD8+ T cells in the lymph node were able to gain cytolytic function over time following infection, but as they did so, lost their ability to remain within the lymph node. These results sparked him to question whether the CD8+ T cells of HIV infected patients remain capable of maintaining a properly functioning lymph node reservoir. When looking at HIV patient PBMCs and lymph nodes, perforin vs. CD27 (a co-stimulatory marker) increased. His lab also saw a decrease in perforin expression in CD8+ T cells in the lymph node after combined anti-retroviral therapy (cART) treatment. Moreover, there was no association between T-bet and perforin in the lymph node. What they did discover about the CD8+ T cells after treatment was that even after entering the lymph node, they still could not produce perforin. Therefore, HIV seems to have an effect on the normal function of CD8+ T cell within the lymph node.
This was a great meeting. Dr Betts talk was very exciting with some great flow data. The next SDSC meeting will most likely be in October. We will keep you posted. We look forward to seeing you there.


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