Multiplex immunofluorescence analysis of pretreatment tumor biopsies. CD86 and PD-L1 are typically expressed by PAX5-positive HRS cells and CD68⁺ macrophages, whereas CTLA-4 and PD-1 are most commonly expressed by CD3⁺ T cells. (A) The densities (cells per mm²) of CD68⁺ macrophages, PAX5(dim)-positive HRS cells, CD86⁺, PD-L1–positive, CTLA-4–positive, and PD-1–positive cells were not associated with a response to ipilimumab monotherapy. (B) Multiplex immunofluorescence staining of pretreatment biopsies from patient T01 (responder, left) and C07 (nonresponder, right) shows that, in both cases, a subset of the PAX5(dim)–positive HRS cells (yellow) coexpressed CD86 (purple) and are surrounded by and in contact with CTLA-4–positive cells (green). Similar expression and distribution of CTLA-4 in responders and nonresponders suggest that they limited efficacy of ipilimumab cannot be attributed to a lack of drug target in the TME.