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Emerging data suggest an important role for T lymphocytes in the pathogenesis of chronic lung disease in preterm infants. Comprehensive assessment of the lymphocyte transcriptome may identify biomarkers and mechanisms of disease.Small volume peripheral blood samples were collected from premature infants enrolled with consent in the Prematurity and Respiratory Outcomes Program (PROP), at the time of discharge from the hospital. Blood samples were collected at two sites and shipped to a central laboratory for processing. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient centrifugation and separated into individual lymphocyte cell types by fluorescence-activated cell sorting. Gating strategies were optimized to ensure reproducible recovery of highly purified lymphocyte populations over a multi-year recruitment period. RNA was isolated from sorted cells and characterized by high-throughput sequencing (RNASeq).Blood volumes averaged 2.5ml, and sufficient PBMCs were collected from 165 of the 246 samples obtained (67%) from the 277 recruited subjects to complete sorting and RNASeq analysis on the resulting sorted cells. The number of total lymphocytes per ml of blood in the neonatal subjects was approximately 4 million/ml. Total lymphocyte frequencies recovered following sort varied widely among subjects, as did the frequency of individual lymphocyte and NK cell sub-populations. RNA yield from sorted cells varied according to cell type, but RNA of sufficient quantity and quality was recovered to enable RNASeq.Our results describe a validated procedure for the generation of genome-wide expression data from isolated lymphocyte sub-populations obtained from newborn blood.

Our previous work has demonstrated that human follicular lymphoma (FL) infiltrating T cells are anergic, in part due to suppression by regulatory T cells. In this study, we identify pericellular adenosine, interacting with T cell-associated G protein-coupled A2A/B adenosine receptors (AR), as contributing to FL T cell hyporesponsiveness. In a subset of FL patient samples, treatment of lymph node mononuclear cells (LNMC) with specific A2A/B AR antagonists results in an increase in IFN-γ or IL-2 secretion upon anti-CD3/CD28 Ab stimulation, as compared with that seen without inhibitors. In contrast, treatment with an A1 AR antagonist had no effect on cytokine secretion. As the rate limiting step for adenosine generation from pericellular ATP is the ecto-ATPase CD39, we next show that inhibition of CD39 activity using the inhibitor ARL 67156 partially overcomes T cell hyporesponsiveness in a subset of patient samples. Phenotypic characterization of LNMC demonstrates populations of CD39-expressing CD4+ and CD8+ T cells, which are overrepresented in FL as compared with that seen in normal or reactive nodes, or normal peripheral blood. Thirty percent of the FL CD4+CD39+ T cells coexpress CD25high and FOXP3 (consistent with regulatory T cells). Finally, FL or normal LNMC hydrolyze ATP in vitro, in a dose- and time-dependent fashion, with the rate of ATP consumption being associated with the degree of CD39+ T cell infiltration. Together, these results support the finding that the ATP-ectonucleotidase-adenosine system mediates T cell anergy in a human tumor. In addition, these studies suggest that the A2A/B AR as well as CD39 are novel pharmacological targets for augmenting cancer immunotherapy.

CD8+ T cells are critically important for immune defense against many viral and bacterial pathogens, and are also key components of cancer immunotherapy. Help from CD4+ T cells is usually essential for optimal CD8+ T cell responses, driving the primary response, the survival of memory cells, and the generation of protective and therapeutic immunity. Understanding the mechanisms of help is thus essential for vaccine design, and for restoring protective immunity in immunosuppressed individuals. Our laboratory has developed an immunization protocol using peptide-pulsed dendritic cells to stimulate help-dependent primary, memory, and secondary CD8+ T cell responses. We have used gene-targeted and T cell receptor transgenic mice to identify two distinct pathways that generate help-dependent and help-independent CD8+ T cell responses, respectively, and are now starting to define the molecular mechanisms underlying these two pathways.

Human peripheral blood dendritic cells (PBDC) are a rare population comprised of several distinctive subsets. Analysis of these cells has been hindered by their low frequency. In this study, we report a novel direct ex vivo 11-color flow cytometric assay that combines subset identification with analysis of activation status and endocytic ability of three major PBDC subsets (CD1c(+)CD11c(+) "MDC1," CD141(+)CD11c(+) "MDC2," and CD303(+)CD11c(-) "PDC") within a single platform. This method eliminates the need for DC enrichment, isolation, or prolonged culture. Human peripheral blood mononuclear cells (PBMC) from healthy donors are incubated with FITC-dextran directly ex vivo, prior to cell surface staining with various markers. As expected, PBDC identified by this assay express low levels of CD40 and CD86 directly ex vivo, and significantly upregulate expression of these molecules upon stimulation with toll-like receptor ligands LPS and CpG oligonucleotides. In addition, PDC internalize FITC-labeled dextran poorly in comparison to MDC1 and MDC2 subsets. Specificity of FITC-dextran endocytosis is further verified by imaging flow cytometry. Furthermore, the combination of surface markers used in this assay reveals a previously unreported CD4(+)CD11c(+)CD303(-)CD1c(-)CD141(-) cell population. Taken together, this assay is a rapid and cost-effective method that avoids manipulation of PBDC while providing direct ex vivo high-dimensional flow cytometry data for PBDC studies.

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.

Recent studies have shown that CD4+ T cell help is required for the generation of memory CD8+ T cells that can proliferate and differentiate into effector cells on Ag restimulation. The importance of help for primary CD8+ T cell responses remains controversial. It has been suggested that help is not required for the initial proliferation and differentiation of CD8+ T cells in vivo and that classical models of helper-dependent responses describe impaired secondary responses to Ag in vitro. We have measured primary CD8+ T cell responses to peptide-pulsed dendritic cells in mice by cytokine ELISPOT and tetramer staining. No responses were detected in the absence of help, either when normal dendritic cells were injected into MHC II-deficient mice or when MHC II-deficient dendritic cells were injected into normal mice. Thus, the primary in vivo CD8+ T cell response depends absolutely on help from CD4+ T cells in our experimental system.

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.

Integrated Raman and angular-scattering microscopy (IRAM) is a multimodal platform capable of noninvasively probing both the chemistry and morphology of a single cell without prior labeling. Using this system, we are able to detect activation-dependent changes in the Raman and elastic-scattering signals from CD8+ T cells stimulated with either Staphylococcal enterotoxin B (SEB) or phorbol myristate acetate (PMA). In both cases, results obtained from the IRAM instrument correlate well with results obtained from traditional fluorescence-based flow cytometry for paired samples. SEB-mediated activation was distinguished from resting state in CD8+ T cells by an increase in the number and mean size of small ( approximately 500-nm) elastic scatterers as well as a decrease in Raman bands, indicating changes in nuclear content. PMA-mediated activation induced a different profile in CD8+ T cells from SEB, showing a similar increase in small elastic scatterers but a different Raman change, with elevation of cellular protein and lipid bands. These results suggest the potential of this multimodal, label-free optical technique for studying processes in single cells.

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 We next examined the proportion of the total CD45RA− memory CD4+ T-cells that were CXCR5+, a phenotype consistent with Follicular B Helper T-cells (TFH). Such TFH cells are crucial for the elicitation of B-cell memory and the generation of high affinity antibody responses. Whereas there was no significant difference in the mean percent of the memory T-cells that have a TFH phenotype in the FLN (64.1±5.0%) as compared to that seen in the RLN (55.8±4.6%), the proportion of TFH in the FLN was significantly higher than that seen in the NLN (48.0±4.0%). As discrete TFH populations have been identified based on their immunophenotype and anatomical localization, we next evaluated the distribution of TFH subpopulations, as defined by their surface expression of CD25 and CD57.TABLE 2Germinal Center (GC)Light zoneOuter zoneDual CD25+CD57+FLN16.4±5.022.5±2.754.4±4.16.72±1.06RLN9.4±0.5*15.5±3.272.9±3.6*2.21±0.50*NLN9.3±1.29.4±1.3*80.0±2.5*1.24±0.13*Statistical analysis was performed using Wilcoxon rank sum tests (distribution free).*p In FLN there appears to be a skewing of TFH subsets away from the CD25−CD57− outer zone subset (a component of which is reported to contain pre-formed CD40L and thus likely provides help to GC-B cells) and towards the CD25+CD57− GC subset (reported to suppress TFH cell help to GC B-cells and inhibit AID expression); and towards the CD25−CD57+ light zone subset (reported to provide help to GC B-cells by inducing expression of AID, as well as inducing immunoglobulin production and class switching). Finally, we identified a potentially unique “dual” CD25+CD57+ population of TFH cells, with a higher frequency seen in FLN (6.72±1.06%) compared to both RLN (2.21±0.50) and NLN (1.24±0.13), with frequencies as high as 12% seen in some FLN samples. Taken together our findings suggest that; (a) the T-cell subset distribution of FL is overall similar to that of RLN but different than that of NLN suggesting that the FLN microenvironment is driven in part by the same immune reactivity and inflammatory signals as seen in RLN and; (b) FLN have a higher proportion of TFH cells then that of NLN, and a different profile of TFH subsets than in both RLN and NLN. Given the critical role that TFH cells play in normal GC-B cell biology, we speculate that differences in the TFH populations in FLN compared to that of RLN and NLN may in part regulate the malignant transformation and/or survival of FL B-cells. Disclosures: No relevant conflicts of interest to declare.

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.