Grote, Deanna M.

Abstract Although extensively studied, the exact mechanism by which TGF-β mediates T-cell inhibition is not entirely clear. When we examined the effect of TGF-β on the phenotype and function of T cells, we noted that CD70 was significantly up-regulated by TGF-β. This surprising finding led us to explore the underlying mechanism by which TGF-β upregulates CD70 expression on T cells and to define the function of CD70+ cells in B-cell NHL. Interestingly, we observed that TGF-β preferentially induced CD70 expression on effector memory T cells while it predominantly up-regulated Foxp3 on naïve T cells. CD70 induction is Smad3-dependent and involves Stat5 signaling. CD70+ T cells were highly susceptible to TGF-β-mediated apoptosis and that transfection of Jurkat cells with the CD70 gene led to a significantly reduced survival. TGF-β-induced CD70+ T cells had a diminished response to immune stimulation associated with downregulation of co-stimulatory molecules CD27 and CD28. CD70-expressing T cells from biopsy specimens of patients with B-cell NHL failed to produce cytokines and displayed no signal transduction when stimulated. CD70-expressing T cells were highly represented in follicular NHL biopsy specimens and increased numbers of CD70+ cells correlated with an inferior patient outcome. These novel findings not only revealed a biological role for a TGF-β/CD70 axis in mediating immune inhibition, but also may lead to therapeutic approaches in patients with B-cell NHL.

Abstract MicroRNAs (miRNAs) are small noncoding RNAs that are approximately 20–22 nucleotides with critical functions in cell growth, survival, and differentiation. These conserved sequences can regulate expression of multiple genes and are often tissue specific and dysregulated in malignancies. Thus, miRNA profiling has been used to create signatures for many solid tumors. These profiles have been used to classify tumors and to help predict survival and outcome. In the present study, we utilized the DiscovArray miRNA profiling service (Asuragen Services, Austin, TX) which utilizes a custom-manufactured Affymetrix GeneChip® from Ambion that covers miRNAs derived from the Sanger miRBase (http://microrna.sanger.ac.uk/sequences/index.shtml) and over 11,000 predicted miRNAs derived from published reports. The signal processing implemented was a multi-step process involving probe-specific signal detection calls, background estimation and correction, constant variance stabilization and global normalization. For each probe, an estimated background value was subtracted derived from the median signal of a set of G-C-matched anti-genomic controls. Arrays within a specific experiment were normalized together according to variance stabilization method. Detection calls were based on a Wilcoxon rank-sum test of the miRNA probe signal compared to the distribution of signals from GC-content matched anti-genomic probes. For statistical hypothesis testing, a two-sample t-test, with assumption of equal variance, was applied. One-way ANOVA was used for multiple group comparison. Probes were considered to be differentially expressed based on two criteria: a p-value of < 0.001 and glog2 difference > 1. miRNA expression was analyzed in all malignant B lineage cells (CD19+ CD138+) (n=8), malignant B cells alone (CD19+) (n=6) and plasma cells alone (CD138+) (n=3) from Waldenström macroglobulinemia (WM) patients. The expression was compared to malignant CD19+ B cells from chronic lymphocytic leukemia (CLL) patients (n=5), malignant plasma cells (CD138+) from multiple myeloma (MM) patients (n=5) and to B lineage cells (CD19+ CD138+) (n=4), CD19+ B-lymphocytes (n=3) and CD138+ plasma cells (n=6) from healthy donors. Data analysis based on a total of approximately 11,000 miRNAs analyzed shows that CD19+ CD138+ cells (double sorting) from WM patients did not cluster as a unique group. Some samples had a pattern similar to CLL, some similar to MM and others similar to CD19+ CD138+ cells from healthy controls. This lack of clear signature was observed by others in gene expression profiling and CGH arrays. We therefore hypothesized this lack of clustering was due to the lymphoplasmacytic nature of WM cells and therefore we analyzed B cells (CD19+) and plasma cells (CD138+) separately. miRNA expression in B cells (CD19+) identifies a signature in normal B cells that is absent in both WM B cells (CD19+) and CLL cells. There is also a set of miRNAs that are absent in normal B cells that are expressed in WM B cells and CLL. In addition, WM B cells had a unique miRNA signature that is unique compared to CLL and normal B cells. An additional set of miRNAs were expressed and clustered only in CLL patients. Similar to B cells, plasma cell (CD138+) analysis in WM, MM and healthy donors shows a clustering pattern that identifies normal plasma cells from MM plasma cells. WM plasma cells had a miRNA signature that is unique only to WM patients, however, a subset of miRNAs shared an expression pattern with MM plasma cells. While miRNAs can target multiple genes, some of the genes that are targets of the miRNAs identified in this analysis include XBP-1, Blimp-1, IRF-4, Bcl-6 and TACI. These target genes are known to be important in B cell and plasma cell development. In summary, we have analyzed miRNA expression in malignant B cells (CD19+) and malignant plasma cells (CD138+) from WM patients and compared their expression pattern to their normal counterpart as well as malignant counterpart in CLL B cells and MM plasma cells. Our analysis shows that WM B cells have a miRNA signature unique to WM only and one that is shared by CLL cells. Similarly, WM plasma cells have a unique miRNA signature but also has some miRNAs that are shared by malignant plasma cells in MM. These miRNAs target genes involved in B cell differentiation. Analysis of the functional roles of these miRNAs will and their regulation will further our understanding of the regulation of B cells development in normal and malignant conditions.

Abstract The tumor microenvironment in B-cell non-Hodgkin’s lymphoma (NHL) plays an important role in supporting the growth and survival of malignant lymphocytes. In contrast, the extent to which this is true in T-cell NHL is less clear. While professional monocytederived antigen-presenting cells (MDC) are required for T-cell activation and survival, a growing body of evidence demonstrates that MDC may promote the survival of malignant T cells. Immunohistochemical staining of skin biopsies from cutaneous T-cell lymphoma (CTCL) patients (n=11) demonstrated a rich infiltrate of CD68+ macrophages and CD1a+ dendritic cells (DC). As these cells are derived from CD14+ monocytes, we examined the role of monocytes in promoting survival in malignant T cells obtained from CTCL patients (n=9). Initially, peripheral blood mononuclear cells (PBMC) were either depleted of CD14+ monocytes (>85% depleted) or mock-depleted prior to being cultured. Viability of malignant CD4+CD7− T cells was determined by Annexin V/7-AAD staining. While 90.9% (S.D. +/− 0.4%) of mock-depleted malignant T cells were viable, only 73.9% (S.D. +/− 1.3%) of monocyte-depleted cells remained viable, suggesting that monocytes may promote the survival of malignant T cells. In a similar fashion, HuT-78 cells (Sézary Syndrome cell line) were cocultured with CD14+ monocytes from normal donors. Compared with HuT-78 cells alone, the presence of CD14+ monocytes resulted in a 2.7 fold increase in thymidine incorporation (n=5; S.D. +/− 0.6). This is largely due to cytokine production, as the same experiment performed with a transwell insert resulted in a 2.1 fold increase in HuT-78 thymidine incorporation (n=5; S.D. +/− 0.4). Similar results were obtained using additional Sézary Syndrome (i.e. SeAx) and Mycosis Fungoides (i.e. MyLa) cell lines. Monocytes obtained from normal donors were subsequently cultured and cell-free conditioned media was obtained 48 hours later. Media supplemented with monocyte-conditioned media (monocyte-CM) prevented the induction of apoptosis in CTCL cell lines as measured by thymidine incorporation, trypan blue exclusion and Annexin V/propidium iodide staining. This affect was monocyte-specific, as neither T-cell nor B-cell conditioned media were observed to promote survival of CTCL cell lines. Furthermore, monocyte-CM had no affect on resting or anti-CD3 activated normal T cells. We next cultured malignant T cells obtained from PBMC (n=6) or skin biopsies (n=3) from CTCL patients in the presence or absence of monocyte-CM and assessed cell viability by Annexin V staining. 32.1% (S.D. +/− 14.7%) and 60.5% (S.D. +/− 21.5%) of malignant T cells isolated from PBMC remained viable in the absence or presence of monocyte-CM, respectively; 13.0% (S.D. +/− 10.8%) of malignant T cells isolated from skin biopsies remained viable, compared with 26.5% (S.D. +/− 8.2%) of cells grown in the presence of monocyte-CM (p=.03). Given the prevalence of MDC within CTCL-involved skin and their potential role in promoting the survival of malignant T cells, one may predict that malignant T cells may recruit monocytes in a chemokinedependent fashion. Therefore, cell-free conditioned media from CTCL cell lines (HuT- 78, SeAx, MyLa) was collected and used to promote monocyte chemotaxis in a standard chemotaxis assay. Only neutralization of CCL5 (RANTES) was capable of abrogating monocyte chemotaxis, demonstrating that the production of CCL5 by malignant T cells promotes monocyte migration. Therefore, we examined serum CCL5 levels by ELISA from both normal donors (n=24) and CTCL patients (n=23) and found an approximately 3-fold elevation in serum CCL5 in CTCL patients (normal donors: mean CCL5 10.0 ng/mL; 95% CI 7.1–13.0; CTCL patients: mean 29.2 ng/mL; 95% CI 20.6–37.8; p<0.0001). Serial serum samples were available for 6 patients, 4 of whom had progressive disease. Serum CCL5 increased from a mean of 17.9 ng/mL before progression (S.D. +/− 7.0) to 44.8 ng/mL (S.D. +/− 20.0) after progression (p=.04). In contrast, serum CCL5 decreased by at least 50% in both patients with responsive disease. Our findings demonstrate that monocytes, recruited by malignant T cells in a CCL5-dependent fashion, promote the survival of lymphoma cells in CTCL, thus raising the possibility that targeting tumor-associated monocytes and MDC may represent a novel therapeutic strategy in CTCL.

Abstract Rituximab is a chimeric murine/human monoclonal antibody that binds to CD20 on B-lymphocytes. Binding of the Fab domain to B-cells directly induces apoptosis, while the Fc domain recruits immune effector functions to mediate cell lysis. Because rituximab therapy alone does not result in durable responses for all patients, combination therapies have been explored. We have previously shown in a phase I study that interleukin-12 (IL-12), which facilitates cytolytic T-cell responses and enhances the lytic activity of NK cells, can be safely combined with rituximab and that IL-12 significantly upregulated gamma interferon, CXCL10 (inducible protein-10) and NK cell activity in the peripheral blood. To confirm whether IL-12 could augment the immune mediated cell lysis induced by rituximab, a subsequent randomized phase II study of the combination was performed in patients with B-cell lymphoma. While the combination of IL-12 and rituximab was well tolerated with acceptable toxicity, only moderate disease activity was seen and the response rate to the combination was similar to that seen with rituximab alone. Additionally, the sequential administration of IL-12 at the time of disease progression after treatment with rituximab did not result in any clinical responses. This study was therefore performed to determine potential biologic reasons for the lack of increased clinical efficacy when IL-12 was added to rituximab therapy in patients with B-cell non-Hodgkin lymphoma. Of the 52 patients treated on the phase II study, 8 patients had matched tumor biopsies and peripheral blood specimens obtained prior to therapy and again 2 weeks after treatment was started. Six of the patients were receiving IL-12 plus rituximab at the time the specimens were obtained while 2 were receiving rituximab alone. Gene expression array analysis using the Affymetrix U133 plus chip was performed on RNA isolated from cells from involved lymph nodes and from peripheral blood mononuclear cells. Specimens from the peripheral blood of patients who received IL-12 in combination with rituximab showed a greater than 5-fold increase in the expression of multiple genes known to be upregulated by IL-12 signaling including interferon gamma, CXCL10, IFIT2 and 4 (interferon-induced protein with tetratricopeptide repeats 2 and 4), IL-8 and CXCL2 (macrophage inflammatory protein-2). These increases in gene expression were not seen in the peripheral blood of patients who received rituximab alone. Furthermore, the significant changes seen in cells obtained from the peripheral blood were not seen in cells obtained from lymph nodes involved by lymphoma, despite the samples being obtained from the same patient on the same day. In the tumor specimens, the changes in gene expression involved none of the genes downstream of IL-12 signaling. Instead, many of the upregulated genes were associated with cell cycle and spindle checkpoint proteins suggesting ongoing tumor cell proliferation. In conclusion, while IL-12 significantly upregulated gene expression in the peripheral blood, the same changes were not seen in the tumor. This would suggest that systemically administered IL-12 may not be effectively delivered to the site of tumor involvement. This finding may explain the lack of additional clinical benefit when IL-12 was added to rituximab as therapy for patients with non-Hodgkin lymphoma.

8538 Background: Follicular lymphoma (FL) is a heterogeneous disease with a variable prognosis. Transformation to an aggressive lymphoma is associated with a poor prognosis. Previous studies have identified tumor microenvironment cells such as CD68+ macrophages, PD-1+ and FOXP3+ cells as prognostic in FL. However, results have been conflicting and infrequently address the risk of transformation. In this study we analyzed various components of the microenvironment and their association with transformation. Methods: Patients with FL that later transformed were identified through Mayo Clinic lymphoma database. Tissue specimens at diagnosis were stained with CD68, CD11c, CD21, CXCL13, FOXP3, PD-1 and CD14 and characterized by pattern of location and semi-quantitative cell content. Time to transformation (TTT) and overall survival (OS) were assessed by Kaplan-Meir analysis. Significant variables were further analyzed by cox proportional hazards model. Results: 58 patients were included with a median TTT of 4.7 years (range, 0.4-20). Presence of CD14+ cells vs absence and their location (follicular vs non-follicular) were associated with shorter TTT (4.5 vs 6.2 yrs., p=0.037 and 3.8 vs 5.9 yrs. p= 0.027, respectively). The quantity of PD-1+ cell content was not associated with TTT or OS. However, localization of PD-1 to the follicle compared to diffuse was associated with a longer TTT (6.1 vs 3.6 yrs. p=0.033) and superior OS (9.7 vs 4.6 yrs. p=0.009). On multivariate analysis, the pattern of CD14 and PD-1 staining remained significantly associated with shorter TTT (Table). Conclusions: In follicular lymphoma, a diffuse pattern of PD-1+ cells is associated with inferior TTT and OS. CD14+ cells localized to the follicle is associated with a shorter TTT. After accounting for FLIPI score, both these factors remained significant. These results identify two independent predictors of the rate of transformation in follicular lymphoma and suggest it is location rather than quantity of CD14+ or PD-1+ cells that influence outcomes. [Table: see text]

Abstract Background - The phenotype and biological role of monocytes in B-cell non-Hodgkin lymphoma (NHL) is not fully understood, however, an increased absolute monocyte count in the peripheral blood of lymphoma patients is associated with a poor prognosis. We have previously reported that monocytes from patients with relapsed B-cell NHL displayed an immunosuppressive CD14+HLA-DRlow/- phenotype that correlated with a poor prognosis. However, the underlying mechanism by which CD14+HLA-DRlow/- monocytes develop in patients with B-cell NHL is unknown. The goal of this study was to determine whether cytokines are responsible for the increased number and phenotype of CD14+HLA-DRlow/-monocytes present in lymphoma patients. Methods – Whole blood from patients with newly diagnosed, untreated B-cell NHL (n=20) and healthy donors (n=20) was stained with a panel of antibodies and analyzed by 10-color flow cytometry. Cytokine levels in serum and culture supernatants were measured by Luminex and ELISA, respectively. Polarization of monocytes from healthy donors was performed by incubating CD14+ cells with specific cytokines or supernatants from B-cell NHL cell lines for 24 hours. Activation and proliferation of CD4+T cells cocultured with IL-10-pretreated monocytes were measured by flow cytometry. Results – By flow cytometry, we observed that the absolute number of peripheral monocytes was increased in newly-diagnosed lymphoma patients compared to healthy donors. A significant proportion of these monocytes displayed an immunosuppressive CD14+HLA-DRlow/- phenotype and the numbers of CD14+HLA-DRlow/- cells were significantly higher in lymphoma patients than in healthy donors. To identify the underlying mechanism by which CD14+HLA-DRlow/- monocytes develop thereby leading to increased numbers of CD14+HLA-DRlow/- monocytes in B-cell NHL, we tested a panel of cytokines and found that IL-10 played a key role in the process. Firstly, treatment of monocytes with IL-10 in vitro resulted in the generation of CD14+HLA-DRlow/- cells. Second, monocytes co-cultured with IL-10 producing lymphoma B-cells, or treated with supernatants from lymphoma cell cultures, developed a CD14+HLA-DRlow/- phenotype. Thirdly, IL-10 levels were increased in the serum of DLBCL (p<0.0001) and FL patients (p=0.010) compared to healthy controls, and serum IL-10 levels correlated with increased numbers of peripheral monocytes (p=0.02). Finally, IL-10-pretreated CD14+HLA-DRlow/- monocytes were significantly immunosuppressive and inhibited the proliferation and activation of CD4+T cells in co-culture assays. Conclusions- Taken together, our results suggest that IL-10 signaling contributes to the increased numbers of CD14+HLA-DRlow/- monocytes in B-cell NHL. Strategies to inhibit IL-10 production may therefore have therapeutic potential in B-cell NHL. Disclosures No relevant conflicts of interest to declare.

Abstract Serum BLyS levels have been found to be elevated in a number of immune disease models and there is increasing evidence that this may correlate with pathogenesis of various B cell related disorders including B cell malignancies. While it is clear that BLyS expression is required for normal B cell development and homeostasis, the exact source of BLyS in these scenarios and definition of the mechanisms that control BLyS expression remain to be fully elucidated. Serum BLyS levels are elevated in a number of B cell malignancies known to have a familial incidence. Therefore, we sought to determine if there was any correlation between serum BLyS levels and family history of B cell cancers. In our initial studies we examined the serum BLyS levels in patients with sporadic vs. familial B-cell chronic lymphocytic leukemia (B-CLL; near relative with B-CLL, multiple myeloma, or non-Hodgkin lymphoma), and normal age-matched controls. In the normal controls (n=50), the mean serum BLyS level was 12.7 ng/ml, and we found that 4/50 (8%) had BLyS levels exceeding 20 ng/ml. In the sporadic CLL cohort (n=52), the mean serum BLyS level was 18.3 ng/ml, and we found that 5/52 (10%) had BLyS levels exceeding 20 ng/ml. In striking contrast, in the familial CLL cohort (n=24), the mean serum BLyS levels was 33.4 ng/ml, and we found that 11/24 (46%) had BLyS levels exceeding 20 ng/ml. The percentage of patients with elevated BLyS levels, as well as the mean BLyS levels in the familial CLL cohort compared to the normal controls, was significantly higher (mean, p=0.002) and suggests that elevated BLyS levels may correlate with familial CLL. Because of the correlation between BLyS levels and family history B cell malignancies, we next wanted to determine if there was a common underlying genetic event influencing BLyS expression in this group of patients. We began by sequencing the BLyS promoter in 19 patients with B-CLL and 11 normal controls. We identified 2 sites that were polymorphic, −661 A/G and −871 C/T. The −871 T/T genotype has been previously reported to be expressed at increased frequency in SLE patients and is associated with elevated BLyS mRNA levels. Using RFLP analysis we examined the presence of the two polymorphisms in our control, sporadic, and familial CLL cohorts. In the control group (n=50) we found that at −871, C/C (wildtype) was expressed in 24%, C/T in 54%, and T/T in 22% of patients. Similarily, in the sporadic CLL group (n=35) we found that at −871, C/C was expressed in 29%, C/T in 57%, and T/T in 14%. In the familial group (n=24) we found that at −871, C/C was expressed in only 8%, C/T in 67%, and T/T in 25%. These data suggest a decreased representation of −871 C/C in the familial group. No significant changes were observed between the three groups at the −661 A/G locus. Additionally, we found that none of the patients with BLyS levels exceeding 20 ng/ml expresses C/C at −871. In summary, our data suggest that BLyS levels are elevated in patients with familial CLL relative to both normal controls and individuals with sporadic CLL and that elevated BLyS levels may correlate with the presence of a T at −871 in the BLyS promoter.

Abstract Background The role of intratumoral PD-1+ T cells and their contribution to patient outcome in follicular lymphoma (FL) has been controversial. While some studies have found that increased numbers of PD-1+ T cells correlate with an inferior prognosis or have no impact on patient outcomes, other studies observed that the numbers of intratumoral PD-1+ T cells are an indicator of a favorable outcome in FL. In previous work, we observed that PD-1 can be expressed by different subsets of intratumoral T cells in FL including T follicular helper (TFH) cells and exhausted T effector cells. However, the function, prevalence, distribution and clinical importance of the respective intratumoral PD-1+ T subsets in FL patients are largely unknown. Goal To characterize PD-1+ T cell subsets and to determine the biological and clinical relevance of these T cell subsets in FL. Results In an analysis of biopsy specimens from 33 previously untreated FL patients, we found that PD-1 is abundantly expressed on intratumoral T cells. A median of 54.9% (range: 17.2-92.6, n=33) of CD4+ or 45.8% (range: 12.8-81.7, n=33) of CD8+ T cells express PD-1 on the cell surface. We found by flow cytometry that PD-1 is expressed on intratumoral CD4+ T cells with bright or dim intensity that represents two different subpopulations in FL. By immunohistochemistry, we identified that CD4+PD-1bright T cells predominantly reside in the lymph node follicles while PD-1dim T cells are mainly located in an inter-follicular pattern. Intratumoral CD4+PD-1bright T cells have a TFH cell phenotype and express CXCR5, secret IL-21 and are BCL-6 positive with no TIM-3 expression. In contrast, CD4+PD-1dim T cells have an exhausted phenotype, express TIM-3 and do not express BCL-6 and CXCR5. Functionally, CD4+PD-1dim T cells displayed reduced proliferation, cytokine production and cell signal transduction. When the CD8+ T cells were analyzed, we found that intratumoral CD8+ T cells, primarily reside in the inter-follicular regions, express PD-1 with low intensity and exhibit similar phenotypic and functional changes to inter-follicular CD4+PD-1dim T cells. These cells express TIM-3 but lack CXCR5 and BCL-6. They also displayed reduced proliferation and cytokine production. Clinically, we observed that both the numbers of CD4+PD-1dim and CD8+PD-1dim exhausted T cells significantly correlate with a reduced progression-free survival in FL patients treated with single agent rituximab (p=0.007 and p=0.04 respectively; n=31). Although it did not reach statistical significance, CD4+PD-1bright TFH cells appeared to correlate with a favorable survival, suggesting that TFH and exhausted T cells differentially impact patient outcome in FL. Conclusion Taken together, these results indicated that PD-1 expression defines two subpopulations with distinct functions that differentially impact patient outcome in FL. This finding provides not only an explanation for the previously discrepant clinical observations, but is also important in the design of future anti-PD-1 antibody-based therapy in FL. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 1586 Transformation growth factor (TGF-β) is a highly pleiotropic cytokine critical to a variety of cellular events such as cell differentiation and apoptosis. TGF-β is synthesized as a prepro-TGF-β precursor and secreted after being processed in Golgi apparatus as a latent form that non-covalently combines both TGF-β and latency-associated protein (LAP). Our previous work in B-cell NHL has shown that the intratumoral T cell composition results in the establishment of a profoundly inhibitory tumor microenvironment. However, the underlying mechanism is only partially understood. In this study, using patient specimens and lymphoma cell lines, we evaluated the role of TGF-β in the tumor microenvironment and determined the effect of TGF-β on the generation of intratumoral TH1, TH17 and Treg cells in B-cell NHL. First, we determined expression of TGF-β and found that a latent form of TGF-β was specifically expressed on the surface of CD19+ B cells, but not on other types of cells from B-cell lymphoma biopsy specimens. By screening cell lines, we found that latent TGF-β was also expressed on the surface of lymphoma cell lines, confirming the finding. Second, we tested whether surface expression by lymphoma cells led to the secretion of TGF-β in culture medium. Using an ELISA assay, we detected variable levels of latent TGF-β in the culture medium of primary malignant B cells (median 100 pg/ml per million cells, range: undetectable −229 pg/ml, n=7). Similarly, lymphoma cell lines secreted variable amounts of TGF-β from undetectable to 200 pg/ml per million cells. Next, we determined the effect of TGF-b on intratumoral T cell proliferation and differentiation. As expected, exogenous addition of TGF-β inhibited the proliferation of T cells. Notably, the proliferation of intratumoral T cells was significantly reduced when co-cultured with lymphoma cells bearing an active form of TGF-β compared to that with lymphoma cells without TGF-β. Using flow cytometry, we showed that the addition of exogenous TGF-β enhanced Foxp3 expression in activated CD4+, CD4+CD45RA+ or CD4+CD45RO+ intratumoral T cells, suggesting that TGF-β promotes the generation of Treg cells in tumor microenvironment. In contrast, TGF-β suppressed the expression of IFN-γ in activated CD4+ T cells and inhibited the up-regulation of IL-12 and IL-23-induced IFN-γ expression in CD4+ cells, indicating that TGF-β suppresses the generation of TH1 cells. TGF-β alone slightly inhibited IL-17 expression in CD4+ T cells; however, TGF-β, in the presence of IL-6 and IL-23, up-regulated IL-17 expression in CD4+ T cells, suggesting proinflammatory cytokines are able to reverse the suppression induced by TGF-β. These results suggest that TGF-β controls the generation of TH1, TH17 and Treg cells contributing to the imbalance of effector TH cells and inhibitory Treg cells in the tumor microenvironment of B-cell NHL. Since malignant B-cells produce TGF-β, these results further support the important role of malignant B cells in the regulation of intratumoral T cell differentiation and the host immune response. Disclosures: No relevant conflicts of interest to declare.

Abstract Waldenstrom’s macroglobulinemia is a serious and frequently fatal illness, however many of the mechanisms leading to this disease are not yet known. It is clear, however, that there is dysregulation of the balance between cell proliferation and programmed cell death. BLyS (B-lymphocyte stimulator) is a newly identified TNF family member expressed by monocytes, macrophages, and dendritic cells. BLyS has been shown to be critical for maintenance of normal B cell development and homeostasis, and has been found to stimulate lymphocyte growth. BLyS is overexpressed in a variety of B-cell malignancies and has been shown to inhibit apoptosis in malignant B-cells. Studies of the effects of BLyS on B cell physiology have shown that it also regulates immunoglobulin secretion. To determine the relevance of the BLyS receptor-ligand system in Waldenstrom’s macroglobulinemia, we examined malignant B cells from 5 patients with Waldenstrom’s macroglobulinemia for their ability to bind soluble BLyS and for the expression of the known BLyS receptors, TACI, BAFF-R, or BCMA. The malignant B cells were found to bind BLyS and express BAFF-R and TACI. BCMA expression was undetectable. We then determined the expression of BLyS in bone marrow specimens from 5 patients with Waldenstrom’s macroglobulinemia by immunohistochemistry and compared it to the expression in 5 normal bone marrow specimens. The lymphoplasmacytic cell infiltrate in the bone marrow of patients with Waldenstrom’s macroglobulinemia showed significantly increased BLyS expression. We further determined the serum BLyS levels by ELISA in stored serum specimens from patients with Waldenstrom’s macroglobulinemia (n=20), and compared them to serum BLyS levels in other patients with lymphoplasmacytic lymphoma without elevated immunoglobulin levels (n=10) and to serum levels in normal controls (n=50). Serum BLyS levels in Waldenstrom’s patients (mean: 49.6ng/ml) as well as those in patients with lymphoplasmacytic lymphoma (mean; 46.7ng/ml) were significantly higher than normal controls (mean 12.6ng/ml). In conclusion, we have demonstrated that malignant B cells from patients with Waldenstrom’s macroglobulinemia express the receptors for BLyS and can bind soluble BLyS. Furthermore, we have found that serum BLyS levels are significantly elevated in patients with Waldenstrom’s macroglobulinemia when compared to controls. Strategies to inhibit BLyS may potentially have significant therapeutic efficacy in Waldenstrom’s macroglobulinemia.