Follicular Lymphoma Infiltrating CD4 + CD25 + GITR + Regulatory T-Cells Are Potent Suppressors of CD3/CD28 Co-Stimulated Autologous and Allogeneic CD8 + CD25 − and CD4 + CD25 − T-Cells.

Regulatory T-cells (Tregs) play a critical role in the inhibition of self-reactive immune responses and as such have been implicated in the suppression of tumor reactive effector T-cells. We demonstrate that follicular lymphoma (FL) T-cells are hypo-responsive to CD3/CD28 costimulation, as assessed by proliferation of CFSE (5-(and-6)-carboxyfluorescein diacetate succinimidyl ester) labeled cells, with only 3.11% ± 2.38 and 2.26% ± 1.76 of the CD8 + and CD4 + T-cells proliferating upon stimulation, respectively (n=7). In contrast, both normal lymph node (NLN), and reactive lymph node (RLN, lymphoid hyperplasia) T-cells proliferate significantly in response to costimulation. Specifically, NLN CD8 + and CD4 + T-cells demonstrate 35.2% ± 31.1 and 18.1% ± 15.9 cells proliferating upon stimulation, respectively (n=7). Similarly, upon stimulation, RLN CD8 + and CD4 + T-cells demonstrate 40.6% ± 22.6 and 40.3% ± 30.3 cells proliferating, respectively (n=5). We identify a population of FL infiltrating CD4 + CD25 + GITR + T-cells that are significantly overrepresented within FL, 9.86% ± 6.70 (n=11) of the CD4 + T-cells, as compared to that seen in NLN, 0.70% ± 0.29 (n=13), or RLN, 1.40% ± 1.04 (n=5). These cells actively suppress the proliferation of autologous nodal CD8 + and CD4 + T-cells after costimulation, as CD25 + magnetic bead depletion of these cells in vitro restores proliferation of the remaining CD25 − T-cells. Specifically, proliferation of FL CD8 + CD25 − and CD4 + CD25 − T-cells increases to 24.05% ± 11.46 and 10.53% ± 6.47, respectively, upon costimulation (n=4). The CD25 + enriched cell fraction contains functionally suppressive cells since add back of unlabelled CD25 + enriched cells to CFSE labeled CD25 − cells results in a decrease in proliferation of the costimulated CD8 + CD25 − and CD4 + CD25 − T-cells, namely 7.59% ± 3.86 and 4.16% ± 1.79, respectively (n=4). These cells also suppress cytokine production (IFN-g, TNF-a and IL-2) from autologous nodal T-cells as assessed by multiplex analysis of culture supernatants. In addition to suppressing autologous nodal T-cells, the FL CD25 + enriched cells are also capable of suppressing proliferation of allogeneic CD8 + CD25 − and CD4 + CD25 − T-cells from NLN as well as normal donor peripheral blood lymphocytes (PBL), regardless of very robust stimulation of the target cells with plate bound anti-CD3 and anti-CD28 antibodies. The allogeneic suppression is not reciprocal, since CD25 + enriched cells derived from either NLN or normal donor PBL, used at the same ratio, are not capable of suppressing allogeneic CD8 + CD25 − and CD4 + CD25 − T-cells derived from FL and in fact, are less suppressive against autologous T-cells than are the FL derived CD4 + CD25 + cells. Whether this is due to a higher proportion of functionally suppressive T cells within the FL derived CD25 + enriched cells, compared to that of NLN or normal donor PBL, or to an increased suppressive capacity of the FL derived CD25 + T cells is currently being investigated. These data show that FL infiltrating CD4 + CD25 + GITR + T-cells have a phenotype and function consistent with Tregs and are very potent suppressors of lymphoma associated-CD8 + and CD4 + T-cells, and therefore may play an important role in lymphoma development, progression and response to treatment.