Analyses of the biologic determinants of TMTV in FL. (A) TMTV (in cubic centimeters), determined by 41% SUV_max, in patients with T-cell^LO or T-cell^RICH infiltrate in the nodes. T-cell infiltrative states were measured by calculating a standardized CD4 and CD8A gene z-score for each sample by multiplex gene hybridization. T-cell^LO: quartiles 1-3; n = 34. T-cell^RICH: quartile 4; n = 11. (B) TMTV, determined by SUV ≥2.5, in patients with T-cell^LO and T-cell^RICH tumors. (C) TLG (in grams), in patients with a T-cell^LO or T-cell^RICH tumor infiltrate. (D) TMTV in patients with a low (FL B cells^LO; n = 27) or high (FL B cells^RICH, n = 27) intratumoral FL B-cell infiltrate. FL B-cell infiltration was quantified by a median cutoff for the percentage of light-chain–restricted CD19⁺ FL B cells (≤60%), by using flow cytometric quantification of tumor lymphocytes in 54 fresh tissue samples. TMTV was determined by the 41% SUV_max method. (E) TMTV in patients with a low Ki67 expression (<20%; n = 23), and high Ki67 expression (≥20%; n = 10). Ki67 expression was assessed by immunohistochemistry, and a 20% cutoff threshold was chosen, as previously published.²⁵ (F) Gating strategy for CD8⁺ T-cell subsets obtained from cryopreserved FL TIL samples to allow for the identification of PD-1⁺LAG3⁻ (activated, ∼58% CD8⁺ cells), PD-1⁺LAG3⁺ (exhausted, ∼18%), and PD-1⁻LAG3⁻ (resting, ∼24%) intratumoral CD8⁺ cell subsets by fluorescence-activated cell sorting (FACS). (G) Gating strategy for intratumoral CD4⁺ T-cell subsets obtained from FL TIL samples to allow for the identification of CXCR5⁺ICOS⁺CD4⁺ T_FH cells, and CD25^HICD127^LOCXCR5⁻CD4⁺ T_REG and CD4⁺ non-T_FH/T_REG subsets within the CXCR5⁻ICOS⁻ population by FACS. (H) Intratumoral CD8⁺ (n = 21) and CD8⁺ cell-subset–specific (n = 14) TCR repertoire clonality, as determined by Simpson’s clonality metric, in patients with a low TMTV (n = 10 for CD8⁺, and n = 8 for CD8⁺ subset-specific cells) and those with a high TMTV (n = 11 for CD8⁺, and n = 6 for CD8⁺ subset-specific cells). A median TMTV cutoff threshold (268 cm³) was used to distinguish a low from a high TMTV (by the 41% SUV_max method). (I) TCR repertoire clonality in patients with a low TMTV (n = 10) and patients with a high TMTV (n = 11) within the sorted intratumoral CD4⁺ cell subsets. The Wilcoxon rank-sum test was used for all analyses.