Interrogating Individual Autoreactive Germinal Centers by Photoactivation in a Mixed Chimeric Model of Autoimmunity

This physiologically relevant chimeric model of spontaneous autoreactive germinal centers allows the linking of cellular localization in vivo with downstream molecular analyses. The main advantage of the mixed chimeric model of autoreactive germinal centers is its versatile and marginal nature, allowing the interrogation of virtually any desired cellular subset or molecular pathway. The physiological relevance of this model of autoimmune disease development enables novel insights that may aid the advancement of new therapies.

This bone marrow chimera model can be used in immunology, immuno-oncology, and stem cell research. The photoactivation component has broad usage, as it links tissue localization to downstream cell analysis. The bone marrow preparation and reconstitution and in vivo labeling and tissue explantation steps are all technically complex procedures that lend themselves well to visual demonstrations.

To extract the donor 564Igi mouse femur and tibia, remove the skin from one hind limb, and pop out the knee and ankle joints by pulling on the foot forcefully. Proceed to break the ankle joint. Then, pull the foot toward the body while holding on to the tibia, thereby stripping the tendons and muscles from the tibia.

Break the knee joint to release the tibia, and pull the bone toward the body while holding on to the femur to strip the tendons and muscles from the femur. Then, make an incision at the hip joint, and cut the tendons before pulling the femur out of the hip socket. After removing the contralateral hind limb bones in a similar manner, carefully rub the bones with a coarse paper towel to remove any remaining muscle and connective tissue, and rinse the stripped bones in ice-cold bone marrow buffer.

Then, use Dumont number seven forceps to transfer the bones to a container of fresh bone marrow buffer on ice. To extract the bone marrow cells, rinse a mortar in ice-cold bone marrow buffer, and use a 10-milliliter serological pipette to replace the wash buffer with 10 milliliters of fresh, ice-cold bone marrow buffer. Use the forceps to transfer the bones to the mortar, and use a pestle to crush and grind the bones to release the bone marrow.

Use the 10-milliliter pipette to collect the bone marrow extract before passing the bone marrow solution through a 70-micrometer cell strainer into a 50-milliliter conical tube on ice. Then, rinse the mortar with an additional 10 milliliters of fresh, ice-cold bone marrow buffer to ensure a complete recovery of the cells. To prepare the donor suspensions, mix the appropriate volumes of photoactivatable GFP and 564Igi donor marrow in a 50-milliliter conical tube, and pellet the cells by centrifugation.

Resuspend the mixed cell pellet at a concentration of one times 10 to the eight cells per milliliter of ice-cold bone marrow buffer, and transfer the cells to a precooled, 1.5-milliliter microcentrifuge tube on ice. Next, confirm a lack of response to toe pinch in the anesthetized CD45.1 recipient mouse, and flick the tube of donor bone marrow cells to ensure an adequate resuspension. Load 200 microliters of bone marrow mix into one 0.3-milliliter, 30-gauge insulin syringe, and, with the recipient on its side, gently stretch the skin above and below the eye to slightly pop the eye out.

Carefully insert the tip of the syringe at an approximate 30-degree angle into the front of the eye socket, taking care to avoid the eye and the surrounding tissue. When the tip of the needle touches the bone underlining the eye socket, retract the needle about 0.5 millimeters before using steady pressure to slowly inject the donor bone marrow. Then, return the mouse to its cage with ad libitum antibiotic water, with monitoring until full recovery.

To label the popliteal lymph node, dilute two microliters of phycoerythrin-labeled rat anti-mouse CD169 antibody in 18 microliters of PBS, and add two 10-microliter droplets of the antibody onto a piece of plastic paraffin film. Load each droplet into a single 0.3-milliliter insulin syringe with a 30-gauge needle, and inject 10 microliters of antibody into the footpad of the anesthetized recipient animal. Before harvesting the lymph node, place a square coverslip on a flat surface, and use a vacuum grease-loaded, five-milliliter syringe to trace the edges of the coverslip with grease about one to two millimeters from each edge.

Transfer the chamber to a cold, flat surface, and fill the vacuum grease chamber with ice-cold bone marrow buffer. To access the popliteal lymph nodes, use straight, fine scissors to make an incision in the skin just below the knee pit of the euthanized recipient animal, and extend the cut upward along the hamstring-line almost to the hip joint. Using Dumont number five or number seven forceps, pull each of the exposed flaps of skin outward to expose the tissue in the popliteal fossa, and use the forceps to carefully enter the fossa just medial to the popliteal vein.

Open and close the forceps along the axis of the leg to expose the underlying popliteal lymph node before using the thumb and index finger to pinch the quadriceps muscle from the front side proximal to the knee, to pop the lymph node out of the fossa. Slide the forceps beneath the lymph node to liberate the node from the surrounding tissue, and place it in the vacuum grease chamber. After the second lymph node has been collected, place a second coverslip onto the vacuum grease rim, and press down gently to close the chamber, taking care to extrude all of the air bubbles.

To identify the germinal centers in harvested spleen tissue samples, place the imaging chamber onto the stage of a two-photon fluorescent microscope, and use a 3.5-milliliter plastic transfer pipette to place a drop of distilled water on top of the upper coverslip. Lower the objective until the point of contact, and use the transmitted light to focus on the top of the tissue. Switch to dark mode and two-photon excitation, and tune the laser to 940 nanometers.

Locate the individual white-pulp areas bordered by the CD169 staining near the surface of the tissue, and identify the periarteriolar lymphoid sheath by the second harmonics generation associated with the central arteriole. In the zone between the periarteriolar lymphoid sheath and the marginal zone, identify the presence of highly autofluorescent, activated tingible-body macrophages and, using these landmarks, draw a region of interest around a single germinal center area. Then, set up a Z-stack of around 100 to 150 micrometers in depth, starting from the surface of the tissue and using a step size of about three micrometers before switching to an 830-nanometer excitation wavelength.

Shut off or dim all of the channels to prevent photodamage to the detectors, and image the stack. Then, switch back to the 940-nanometer excitation wavelength, and reopen the channels, scanning through the stack to confirm an efficient photoactivation and an absence of photodamage throughout the image stack. Serotyping of the mixed bone marrow chimeras reveals normalized B cell numbers at six weeks post-reconstitution, with a low frequency of 9D11-positive circulating B cells derived from the 564Igi compartment.

Within the total lymphocyte gate, there was a low frequency of residual recipient-derived cells, about 6%of which are CD45.1-derived, indicating an overall degree of chimerism of about 94%There was a virtually complete chimerism in the B cell compartment and a dominance of photoactivatable GFP bone marrow-derived B cells, a consequence of the heavy negative selection of 564Igi-derived B cells. As observed, in vivo labeling with CD169 facilitates a robust visualization of the marginal zone. The second harmonics signal is apparent in collagen-containing structural elements and major vessels, including the central arteriole of the periarteriolar lymphoid sheath and highly autofluorescent, activated tingible-body macrophages associated with the germinal center activity.

Taken together, these data allow the identification of a region of interest that likely contains a single, photoactivatable germinal center. Downstream flow cytometric evaluation further confirms the presence of normalized B cell compartment numbers, a spontaneous germinal center population, and a subset of photoactivated germinal center B cells. It is important to restrict the total turnaround time of explanting, photoactivation, and tissue processing steps to four to six hours to ensure a sufficient cell viability.

The photoactivated cells can be flow-sorted and subjected to single-cell sequencing to, for instance, characterize the B cell receptor repertoire of a single germinal center. The mixed chimera model has allowed the exploration of autoreactive germinal center biology at a new depth, for example, providing insight into how the process of epitope spreading unfolds. Note that the light source for two-photon microscopy are very powerful pulsed Class 4 lasers that can severely damage the eyes.