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Follicular lymphoma (FL) is one of the most immune responsive of all human malignancies. However, immunoregulatory mechanisms in the tumor microenvironment may impair the efficacy of endogenous immunity as well as therapeutic vaccines and adoptive T-cell therapy. CD4+CD25+CD127loFoxp3+ regulatory T cells (Tregs) are one of the most potent suppressors of effector T cells and were recently shown to be increased in peripheral blood and tumors of patients with various malignancies. Here, we determined the percentage of Tregs, in the tumors and the peripheral blood mononuclear cells (PBMC) of patients with FL (n = 13) at the time of initial diagnosis and after induction of clinical remission with a uniform cyclophosphamide and doxorubicin containing chemotherapy regimen, PACE (Modified ProMACE without Methotrexate; Prednisone, Doxorubicin, Cyclophosphamide, and Etoposide). We observed that Tregs are markedly increased in number both in the tumors (median 19.45%, range 13.4%–44.5%) and PBMC (median 17.7%, range 7.9%–64.5%) of patients with FL at the time of initial diagnosis as compared with PBMC from normal donors (median 3.33%, range 1.88%–6.3%). Unexpectedly, Tregs persisted in high numbers in peripheral blood after induction of clinical remission with a cyclophosphamide and doxorubicin containing chemotherapy regimen (median 19.95%, range 7.6%–67.2%). To determine the immunosuppressive effects of Tregs, we assessed the proliferation of CFSE-labeled effector T cells following polyclonal stimulation with anti-CD3 and anti-CD28 antibodies. The presence of high numbers of Tregs was associated with significantly decreased proliferation of peripheral blood CD4+ T cells in patients with FL both at baseline (median 24.9%, range 3.7%–40.7%) and after chemotherapy induced clinical remission (median 15.8%, range 1.5%–23.5%) as compared with normal donors (median 38%, range 11.7%–58.8%). A similar decrease in proliferation was also noted in CD8+ T cells in the presence of high numbers of Tregs both at baseline and after induction of clinical remission in patients with FL as compared with normal donors. More importantly, intratumoral and peripheral blood Tregs significantly inhibited effector T cells proliferating in response to autologous tumor cell stimulation. In conclusion, these results suggest that CD4+CD25+CD127loFoxp3+ Tregs that inhibit the function of tumor-specific effector T cells are markedly increased in number in both tumor and peripheral blood of patients with FL and persist in high numbers despite induction of clinical remission with standard cyclophosphamide and doxorubicin containing chemotherapy. This observation may potentially explain the failure of two recent Phase III therapeutic vaccine trials in patients with FL. Furthermore, these results suggest that a state of peripheral tolerance continues to be present after induction of clinical remission and strategies that deplete or block function of Tregs may be necessary to enhance the efficacy of therapeutic vaccines and adoptive T-cell therapy in patients with follicular and possibly other non-Hodgkin’s lymphomas.

Follicular lymphoma is one of the most immune-responsive of all human malignancies. However, immunoregulatory mechanisms in the tumor microenvironment may impair the efficacy of immunotherapies such as vaccines and adoptive T-cell therapy. The inhibitory receptor programmed death 1 (PD1), a negative regulator of activated T cells was recently shown to be upregulated on the surface of HIV-specific CD4+ and CD8+ T cells in humans and was associated with impaired T-cell function. Blockade of the immunoregulatory PD-1/PD-ligand 1 (PD-L1) pathway with antibodies against the PD-L1 augmented the function of HIV-specific CD4+ and CD8+ T cells (Day CL et al, Nature, 2006). To investigate the role of PD-1 in lymphoma, we examined PD-1 expression on peripheral blood mononuclear cells (PBMC) and intratumoral T cells in patients with follicular lymphoma prior to therapy. We observed that PD-1 expression is significantly upregulated on peripheral blood and intratumoral CD4+ and CD8+ T cells in patients with follicular lymphoma as compared with normal donor PBMC. Furthermore, PD-1 expression was significantly higher on intratumoral (mean 61%, range 34% to 86%) compared with peripheral blood CD4+ T cells (mean 25%, range 9 to 40%). Likewise, PD-1 expression was significantly higher on intratumoral (mean 44%, range 31% to 69%) compared with peripheral blood CD8+ T cells (mean 16%, range 9 to 31%). PD-1 expression on CD4+ and CD8+ T cells was associated with impaired type 1 cytokine production (IL-2, TNFa, and IFNg) and blockade of the PD-1/PD-ligand pathway with antibodies against PD-1 significantly enhanced T-cell function. These data indicate that the immunoregulatory PD-1/PD-ligand pathway is operative in patients with follicular lymphoma. Moreover, this pathway of reversible T-cell impairment provides a potential target for enhancing the function of exhausted T-cells in follicular lymphoma in combination with other immunomodulatory strategies such as vaccines and adoptive T-cell therapy.