Likewise, the other immune checkpoint markers—including B7-H3, B7-H4 (tested and observed in MCs from the lung cancer tissue microarray positive control), VISTA, LAG3, ICOS, and TIM3—and the immune checkpoint stimulatory markers such as IDO-1 and OX40—were observed in different immune cells with similar distribution patterns between IHC and mIF in tonsil and other control tissues. The gene discussed is ICOS; the disease is lung cancer.