T follicular helper cells are newly identified CD4 T cells subset that provide help to B cells in high affinity animal production. Demonstrating the identification of Tfh cells in an influenza virus infection will help new researchers understand how to perform the necessary techniques for Tfh-associated studies. Because an inappropriate virus titer can cause mortality or a too weak immune response, we advise the titer to be determined before performing an experiment.
For PR8 virus inoculation of eight-week-old male C57 black 6 mice. Under biosafety level 2 conditions, thaw a minus 80-degree Celsius stored PR8 virus stock on ice. Vortex the virus to obtain a homogenous solution.
Dilute the virus to two platforming units per microliter of PBS in a pre-chilled 1.5-milliliter tube and confirm a lack of response toe pinch in the anesthetized animals. Vortex the virus again. Load a 20-microliter pipette tip with 10 microliters of virus.
Carefully add drops of virus into one naris of up to three sedated animals before repeating the inoculation on the other side. Then, place all of the mice in sternal recumbency in warm cages with monitoring until full recovery, and weigh each mouse daily for 10 days. At the end of the experiment, place the mice into a biosafety level 2 laminar flow hood, and secure the first animal to an absorbent, paper-covered foam dissection board.
Use dissection scissors to cut the skin along the abdominal midline, stretching the skin with tweezers and pinning the skin to the dissection board. Open the peritoneum to collect the spleen, and place the spleen into a 70-micrometer filter with a 6-centimeter dish containing 5 milliliters of DMEM supplemented with 1%FBS. Split the diaphragm and and ribs up to the thymus and secure the rib to the side to expose the thoracic cavity.
Move the lung and heart to locate the mediastinal lymph node near the ventral side of the trachea. Then, place the lymph node in its own strainer in a second 6-centimeter dish containing 5 milliliters of medium and use 3-milliliter syringe plungers to gently mash both tissues through the mesh of both strainers. Rinse each strainer with 1 milliliter of fresh DMEM plus FBS, and transfer the cell suspensions to individual 15-milliliter centrifuge tubes.
Collect the cells by centrifugation and thoroughly resuspend the pellets in 1 milliliter of fresh DMEM plus FBS per tube. Then add four additional milliliters of medium to the spleen cell suspension and place both tubes on ice. To stain the cells for CXCR5 expression, invert the tube several times to mix and transfer 2 times 10 to the 6 cells per sample into individual FACS tubes.
Add 2 milliliters of staining buffer to each tube and mix the cells by vortexing. Pellet the cells by centrifugation and discard the supernatants. Gently tap each tube on absorbent paper twice and gently flick the two bottoms to resuspend the cells in the residual supernatant.
Block the nonspecific binding with 0.2 microliters of Fc receptor block per tube, and flick the tube gently to mix before placing the tubes on ice for 10 minutes. At the end of the incubation, add 0.3 microliters of biotin anti-mouse CXCR5 to each tube and mix by gentle flicking. Place the tubes on ice for one hour with gentle flicking at 30 minutes.
At the end of the incubation, add 2 milliliters of staining buffer to the tubes and vortex to mix before sedimenting the cells by centrifugation. Discard the supernatant and flick to resuspend the cells in the residual buffer. Then, place the tubes on ice and label the cells with antibodies against the surface marker of interest as outlined in the table as just demonstrated.
At the end of the incubation, wash the cells with 2 milliliters of staining buffer and resuspend each pellet in 400 microliters of fresh staining buffer for analysis by flow cytometry. PR8 virus-infected mice begin to lose weight on day six, reaching the highest loss on day eight and returning to initial weight levels on day 10. As expected, weight loss is not observed in PBS-treated control mice.
Virus infection also leads to a robust lymphocyte expansion within the draining lymph node. As observed by flow cytometric analysis, T follicular helper cell differentiation initializes at day five and peaks at day 10 post infection with robust numbers of T follicular helper cells induced in influenza virus-infected mice compared to control animals. Further, in both mediastinal lymph nodes and spleens, influenza antigen-specific CD4-positive T cell numbers are induced significantly compared to those observed in control mice with increased populations of PD-1 positive CXCR5-positive cells in PR8 animals.
In addition, intracellular staining for IL-21 reveals that PR8 infection induces significantly higher production of this cytokine in response to virus infection. Immunofluorescent staining indicates the presence of an induced germinal center reaction in PR8-infected mice, and ELISA demonstrates the generation of influenza virus-specific antibodies in virus-infected animals. It is important to perform the CXCR5 staining on ice for an hour, as the quality of the stain will be affected by the staining temperature and the time.
Although we demonstrated CXR5 and IL-21 staining, other surface markers, transcription factors, and cytokines can be assessed by this method to provide multitudes about Tfh differentiation and function.
