Abstract
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.
Separation of subpopulations of rat lymph node cells cytotoxic for tumour target cells and capable of inhibiting cytotoxicity in vitro
