ALBERT

Description: Abstract 2948 Poster Board II-924 The tumor microenvironment of follicular lymphoma (FL) has been shown to play a critical role in the biology of this disease, and to be predictive of treatment outcome for FL patients. To gain further insight into the biology of the FL microenvironment, we asked whether differences exist between the T-cell populations within FL involved lymph nodes (FLN; n=13), as compared to that seen in either reactive (RLN; n= 10) or normal lymph nodes (NLN; n=11; obtained from patients undergoing vascular surgery whereby lymph nodes are removed to gain access to the vascular structures), using 11-color flow cytometry. Interestingly, the proportions of the T-cell populations shown in Table 1 were not statistically different between FLN and RLN, whereas they were statistically different between FLN and NLN. Specifically, the FLN demonstrate higher proportions of CD4+CD45RA−CCR7− T-effector memory cells (TEM) and lower proportions of both CD4+CD45RA−CCR7+ T-central memory (TCM) and CD4+CD45RA+ naïve T-cell populations, as compared to that of the NLN. In addition, within the FLN the TCM subpopulations demonstrate a higher proportion of CXCR5+ non-polarized T-cells as compared to the NLN. In contrast, when the proportions of the TEM subpopulations were examined, there were no significant differences between the FLN, RLN and NLN.TABLE 1CD8+ (%CD3+)CD4+ (%CD3+)Naïve (%CD4+)TCM (%CD4+)TEM (%CD4+)non-preTH1 (%TCM)non-polarized (%TCM)pre-TH1 (%TCM) FLN18.2±2.570.8±3.215.0±2.115.5±1.764.0±3.030.5±3.749.2±5.020.2±2.2 RLN14.4±2.879.9±3.423.6±5.215.0±2.452.6±5.839.8±3.435.4±3.224.3±3.2 NLN10.5±1.4*82.9±2.1*28.7±4.7*21.9±1.345.4±4.9*39.0±1.9*34.2±3.0*25.8±1.7Statistical analysis was performed using Wilcoxon rank sum tests (distribution free).*p

Description: The follicular lymphoma (FL) T-cell microenvironment plays a critical role in the biology of this disease. We therefore determined the lineage, differentiation state, and functional potential of FL-infiltrating CD4+ T-helper cells (TH) compared with reactive and normal lymph node (NLN) TH cells. Relative to NLNs, FL cells have decreased proportions of naive and central memory but increased proportions of effector memory TH cells. We further show differences in the distribution and anatomical localization of CXCR5+ TH populations that, on the basis of transcription factor analysis, include both regulatory and follicular helper T cells. On Staphylococcus enterotoxin-B stimulation, which stimulates T cells through the T-cell receptor, requires no processing by APCs, and can overcome regulator T cell-mediated suppression, the proportion of uncommitted primed precursor cells, as well as TH2 and TH17 cells is higher in FL cells than in reactive lymph nodes or NLNs. However, the proportion of TH1 and polyfunctional TH cells (producing multiple cytokines simultaneously) is similar in FL cells and NLNs. These data suggest that, although TH-cell differentiation in FL is skewed compared with NLNs, FL TH cells should have the same intrinsic ability to elicit antitumor effector responses as NLN TH cells when tumor suppressive mechanisms are attenuated.

Description: Abstract 3119 Tumor infiltrating T-cells tend to be hypo-functional and this loss of function may be due to intrinsic T-cell defects, impaired antigen (Ag) presentation, and/or suppression induced by extrinsic components of the microenvironment, such as regulatory T-cells (Tregs). Each of these potential mechanisms has distinct implications on the potential efficacy of immunotherapy. To determine the functional potential of follicular lymphoma (FL) derived T-cells, we analyzed, by flow cytometry, T helper (Th) subsets and Staphylococcus enterotoxin B (SEB)-induced cytokine profiles of single cell suspensions from FL involved nodes (FL; n=8), reactive lymph nodes (RLN; n=7) and normal lymph nodes (NLN; n=6; obtained during vascular surgery). SEB was used as it directly triggers the T-cell receptor, abrogating the need for Ag presentation, and overcomes Treg mediated suppression. Herein we show that, relative to NLN, FL has decreased proportions of CD4+ T-cells having either a naïve (CD45RA+) or central memory (CD45RA−CCR7+) phenotype but increased proportions of effector memory T-cells (CD45RA−CCR7−). In addition, a higher percentage of pre-stimulation FL CD4+ T-cells show an activated (CD69+) phenotype as compared to that of RLN or NLN. Upon SEB stimulation, the FL CD4+ T-cells, like those from RLN and NLN, show an additional increase in the proportion of CD69+ cells, demonstrating that the FL derived CD4+ T-cells can be activated even further. We also show that upon stimulation with SEB; (a) the proportion of Th1 cells (IL-2+IFN-g+IL-4−) in FL is similar to that seen in RLN or NLN; (b) in contrast, we observe an increased frequency of primed uncommitted precursor Thpp cells (IL-2+IFN-g−IL-4−) in FL compared to that seen in either RLN or NLN; (c) an increased proportion of Th2 cells in FL compared with NLN and; (d) an increase in the proportion of Th17 cells in FL compared to that in RLN. Lastly, the proportions of FL Th cells producing 3 or 4 cytokines simultaneously, or poly-functional CD4+ T-cells, (PFT; PFT-3 producing IL-2, IFN-g and TNF-a or PFT-4 producing IL-2, IFN-g, TNF-a and MIP-1b), after SEB stimulation is similar to that seen in RLN or NLN. These data suggest that although there is skewed Th cell differentiation in FL, as compared to that of RLN or NLN, the intrinsic ability of the FL Th cells to elicit a clinically relevant effector response (both a Th1 and Th2 response) is fully preserved. In addition, the retention of effector function of FL Th cells is further supported by the fact that the proportions of these Th cells that have poly-functional cytokine profiles after SEB stimulation is similar in FL as compared to RLN or NLN. Indeed, poly-functionality of Th cells has been shown to correlate with the elicitation of protective immunity after vaccination for infectious diseases. Finally, the proportion of uncommitted Thpp cells after SEB stimulation is highest in FL. Thpp cells are non-polarized and can still differentiate into either Th1 or Th2 cells. They can also produce several chemokines and thus may play a role in shaping the FL microenvironment by recruiting other immune-effector cells as well as developing into Th1 and Th2 cells. Taken together, our data shows that FL Th cells are fully functional within the parameters of our assays, suggesting that these cells are intrinsically capable of mediating effective anti-tumor immune responses after immunotherapy. Therefore the hypo-functionality of FL T-cells is likely due to extrinsic factors which suppress T-cell function in vivo. Thus the challenge is to develop immunotherapeutic strategies that overcome these tumor associated extrinsic mechanisms, resulting in effective anti-tumor immunity. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 758 The immunosuppressive nature of the tumor microenvironment may limit the efficacy of immunotherapy for patients with follicular lymphoma (FL). We previously showed that FL infiltrating T-cells (FL-T) are hypo-responsive to potent stimulation with plate bound anti-CD3/anti-CD28 antibodies as determined by proliferation and cytokine production. We showed that one mechanism for hypo-responsiveness was suppression by infiltrating CD4+CD25HIGITR+ regulatory T-cells (Hilchey et al. J. Immunol. (2007) 178:4051-61). Here we identify an additional novel mediator of FL-T hypo-responsiveness: pericellular adenosine, generated through the hydrolysis of extracellular ATP, and mediating its effects through interaction with FL-T associated A2A/B adenosine receptors (AR). We show that FL-T hypo-responsiveness is attenuated in a subset of FL patient samples by blocking the A2A/B, but not the A1 AR. Specifically, treatment of lymph node mononuclear cells (LNMC) derived from FL biopsy specimens with the specific A2A/B AR antagonist SCH58261 results in an increase in IFN-g and/or IL-2 secretion upon soluble anti-CD3/anti-CD28 antibody stimulation (in 3 of 6 patient samples tested), whereas treatment with an A1 AR antagonist, CPX, had no effect on cytokine secretion. In contrast to that seen with FL LNMC, SCH58261 treatment of stimulated LNMC derived from normal lymph nodes (NLN; n=5) had no effect on IFN-g and/or IL-2 secretion. As the rate limiting step for adenosine generation from pericellular ATP is the ectoATPase CD39, we next determined the effect of inhibiting CD39 activity on stimulated FL-T cytokine production. Inhibiting CD39 function with the selective CD39 antagonist ARL 67156, partially overcomes FL-T hypo-responsiveness in a subset of patient samples (2 of the 5 patient samples tested). Given the pivotal role that CD39 plays in the ATP-CD39-adenosine-A2AR pathway, we next determined whether the frequency of CD39 bearing T-cell populations differed in FL nodes as compared to that seen in NLN, reactive lymph nodes (RLN), or normal donor peripheral blood (PBMC), using 12 color flow cytometry. We show that CD4+CD39+ and CD8+CD39+ T-cell populations are overrepresented in FL as compared to that seen in NLN. In addition, 30% of the FL CD4+CD39+ T-cells have a regulatory T-cell (Treg) phenotype, co-expressing CD25HI and FOXP3 suggesting that the increased numbers of CD4+CD39+ T-cells observed within FL, as compared to NLN or RLN, are accounted for in part by the increased numbers of Tregs infiltrating the FL. Finally we show that the FL and NLN T-cell associated CD39 is functional, as it hydrolyzes ATP in vitro, in a dose and time dependent fashion. Taken together, the data suggest that the ATP-CD39-adenosine-A2AR pathway is one mechanism for T-cell hypo-responsiveness in FL. Indeed, to our knowledge, this is the first demonstration that this pathway is immunologically significant in any human tumor. The data also suggest that pharmacological inhibition of CD39 ecto-ATP-diphosphohydrolase activity, and/or blockade of the adenosine-A2AR interaction may be rationale strategies to enhance the efficacy of immunotherapeutic treatment approaches for patients with FL. Disclosures: No relevant conflicts of interest to declare.