Follicular lymphoma (FL) is an indolent B cell lymphoma with a heterogenous disease course, and patients may not require immediate treatment upon diagnosis. Scrutiny of its microenvironment may provide key insights into lymphomagenesis and enhancement of therapeutic options. We analyzed the T-cell composition of a large, well-annotated follicular hyperplasia (FH
n=43) cohort utilizing standardized high dimensionality flow cytometry (>
150,000 cells analyzed/sample) and a novel reproducible analytical pipeline leading to identification of even minor T-cell subsets. This baseline reference set was compared to prospectively collected FL samples (n=91) from untreated patients (FL-UT) and patients with relapsed/refractory disease (FL-RR). Compared to FH, both FL-UT and FL-RR specimens exhibited depletion of CD4+ and CD8+ naive subsets and were characterized by an immune suppressive microenvironment enriched in specific inhibitory T-cells, along with exhausted memory T-cells overexpressing varying combinations of immune checkpoint receptors. FL specimens showed enrichment of T follicular regulatory cells (TFR) and two highly suppressive regulatory T-cell (Treg) populations expressing TIGIT and CTLA4 (TC) and PD1, TIGIT, CTLA4, and TIM3 (PTCTi). FL-UT cases with either increased T-reg TC or increased T follicular helper cells (TFH) showed reduced time to first treatment (.