At Dr. Sanz’s lab, this immunomics technique has been used to understand the role of plasmablasts in flaring SLE patients, for example.
Next, researchers isolate the B cells, and study the transcriptome and antibody repertoire, said Dr. Sanz. Conventional, six-color flow cytometry only shows a few naive B cell populations: memory cells, plasmablasts or double-negative (DN) cells. When he and his young sons decorated a sidewalk with colored chalk, he realized more colors were necessary. By using 12-color cytometry, “we can now identify up to 29 populations of B cells that are very reproducible and discrete, and you can identify how they may be dysregulated in SLE,” he said. Double-negative B cells, including DN1 and DN2, are intriguing in lupus pathogenesis. “Critical insights can come from unexpected sources.”
‘You can deplete the B cells of a group of patients who are B cell dependent, & they will do well. The challenge is to recognize those patients & then achieve that degree of depletion.’ —Ignacio Sanz, MD
Dr. Sanz and his team use 12-color cytometry in an ongoing study of 135 SLE patients whose B cell profiles are compared to 25 healthy controls. Profiles of one group of SLE patients, those with a loss of unswitched memory cells and expansion of newly defined activated naive cells and DN2 cells, look very different from other lupus patients with B cell profiles quite similar to those of healthy individuals. The first subset of patients tend to have higher disease activity and include a high concentration of African Americans, more nephritis and skin disease, high anti-Smith, high anti-RNP and high interferon-α, he said. The profiles are then mapped to measure the distance from a mean of normal B cell activity in healthy people. Patients who map farthest from the normal mean flare faster, more often and more intensely, he said.
Dr. Sanz said, “We believe this means there’s probably a B cell-dependent disease and a B cell-independent disease.” Patients with dependent disease may flare more severely and respond better to B cell therapies, but the opposite is true for those with independent disease. Using this mapping exercise, they also explored B cell abnormalities in chronic cutaneous lupus and explored patients’ risk for progression to SLE.3
Immunomics may help identify specific molecular targets or pathways for subsets of SLE patients that lead to innovative clinical trials with mechanistic endpoints that reveal both responders to treatment and nonresponders, who are just as important to understand, said Dr. Sanz.
Double-Negative Cells
The B cell pathway may be split into two compartments: germinal cell-derived with long-lived memory cells and plasma cells, and then an extrafollicular pathway that derives from new activation of autoreactive, naive cells that activate and generate short-lived plasmablasts, he said. This second pathway is very active in lupus flares. Double-negative B cells are highly prevalent in the peripheral blood in some patients, mostly African Americans with active lupus, and usually coincide with large expansions of plasmablasts. Lupus patients with a large expansion of DN2 cells also have high levels of CD19, and lack the follicular marker CXCR5. DN2 cells are also prominent in cohorts of African American SLE patients.
