Transforming growth factor beta (TGF-β) plays an important role in mediating T-cell suppression in B-cell non-Hodgkin lymphoma (NHL). However, the underlying mechanism responsible for TGF-β-mediated inhibition of effector memory T (Tm) cells is largely unknown. As reported here, we show that exhaustion is a major mechanism by which TGF-β inhibits Tm cells, and TGF-β mediated exhaustion is associated with upregulation of CD70. We found that TGF-β upregulates CD70 expression on effector Tm cells while it preferentially induces Foxp3 expression in naïve T cells. CD70 induction by TGF-β is Smad3-dependent and involves IL-2/Stat5 signaling. CD70+ T cells account for TGF-β-induced exhaustion of effector Tm cells. Both TGF-β-induced and preexisting intratumoral CD70+ effector Tm cells from B-cell NHL have an exhausted phenotype and express higher levels of PD-1 and TIM-3 compared to CD70− T cells. Signaling transduction, proliferation and cytokine production are profoundly decreased in these cells and they are highly susceptible to apoptosis. Clinically, intratumoral CD70-expressing T cells are prevalent in follicular B-cell lymphoma (FL) biopsy specimens, and increased numbers of intratumoral CD70+ T cells correlate with an inferior patient outcome. These findings confirm TGF-β-mediated effector Tm cell exhaustion as an important mechanism of immune suppression in B-cell NHL.

Background: Non-Hodgkin lymphomas (NHL) are increasing in incidence and are now the fifth most common tumor diagnosed each year in the United States. Most NHLs are of B-cell origin but the tumor tissue is variably infiltrated with T-cells. Our group has shown in diffuse B-cell large cell lymphoma, that a high number of intratumoral CD4+ T-cells predicts a better overall survival. It has been shown that recently-characterized CD4+CD25+ regulatory T-cells (Treg cells) played an important role in the mediation of anti-tumor immunity. However, there is no data on the role of intratumoral Treg cells in suppression of autologous infiltrating CD4+ T-cells in B-cell NHL. Goal: To investigate the effect of intratumoral Treg cells on the proliferation of tumor-infiltrating CD4+CD25- T-cells, to determine the underlying mechanism of the T-cell suppression, and evaluate the role that malignant B-cells may play in the recruitment of Treg cells to the site of B-cell NHL. Results: We identified a subset of CD4+CD25+ T-cells over-represented in biopsy specimens of B-cell NHL (these cells comprise 17% of cells in lymphoma biopsies, compared 7% of peripheral blood mononuclear cells, 12% of cells in inflammatory tonsil and 6% of cells in tumor free lymph nodes; p-value =0.001). These CD4+CD25+ T-cells are memory-like T-cells (CD45RO+ and CD45RA−) and express high levels of CTLA-4 and Foxp3 when compared to autologous tumor-infiltrating CD4+CD25- T-cells. Importantly, these CD4+CD25+ T-cells displayed the ability to suppress the proliferation and cytokine (IFN-g and IL-4) production of tumor-infiltrating CD4+CD25- T-cells in response to PHA stimulation. Treatment with anti-B7-H1 antibody or PD-1 fusion protein enhanced the proliferation of infiltrating CD4+CD25- T-cells when co-cultured with intratumoral CD4+CD25+ T-cells. Our results suggest that interaction between B7-H1 and PD-1 accounts for about 30% of intratumoral Treg cell-mediated inhibition of autologous infiltrating CD4+CD25- T-cells in tumor sites of B-cell NHL. Lastly, we found that CCL22 secreted by lymphoma B-cells is involved in the chemotaxis and migration of intratumoral CD4+CD25+ T-cells which express chemokine receptor CCR4, but not CCR8. Conclusion: Our results suggest that tumor microenvironmental CD4+CD25+ regulatory T-cells are important regulators of tumor immunity and that these cells are recruited to the area of lymphoma involvement by the malignant B-cells.

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The present study hypothesized that superoxide (O[Formula: see text]·) importantly contributes to the regulation of hypoxia-inducible factor (HIF)-1α expression at posttranscriptional levels in renal medullary interstitial cells (RMICs) of rats. By Western blot analysis, it was found that incubation of RMICs with O[Formula: see text]· generators xanthine/xanthine oxidase and menadione significantly inhibited the hypoxia- or CoCl2-induced increase in HIF-1α levels and completely blocked the increase in HIF-1α levels induced by ubiquitin-proteasome inhibition with CBZ-LLL in the nuclear extracts from these cells. Under normoxic conditions, a cell-permeable O[Formula: see text]· dismutase (SOD) mimetic, 4-hydroxyl-tetramethylpiperidin-oxyl (TEMPOL) and PEG-SOD, significantly increased HIF-1α levels in RMICs. Two mechanistically different inhibitors of NAD(P)H oxidase, diphenyleneiodonium and apocynin, were also found to increase HIF-1α levels in these renal cells. Moreover, introduction of an anti-sense oligodeoxynucleotide specific to NAD(P)H oxidase subunit, p22 phox , into RMICs markedly increased HIF-1α levels. In contrast, the OH· scavenger tetramethylthiourea had no effect on the accumulation of HIF-1α in these renal cells. By Northern blot analysis, scavenging or dismutation of O[Formula: see text]· by TEMPOL and PEG-SOD was found to increase the mRNA levels of an HIF-1α-targeted gene, heme oxygenase-1. These results indicate that increased intracellular O[Formula: see text]· levels induce HIF-1α degradation independently of H2O2 and OH· radicals in RMICs. NAD(P)H oxidase activity may importantly contribute to this posttranscriptional regulation of HIF-1α in these cells under physiological conditions.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a transcription factor that regulates the oxygen-dependent expression of a number of genes. This transcription factor may contribute to the abundant expression of many genes in renal medullary cells that function normally under hypoxic conditions. The present study was designed to determine the characteristics of HIF-1alpha cDNA cloned from the rat kidney and the expression profile of HIF-1alpha in different kidney regions and to explore the mechanism activating or regulating HIF-1alpha expression in renal medullary cells. A 3,718-bp HIF-1alpha cDNA from the rat kidney was first cloned and sequenced using RT-PCR and TA cloning technique. It was found that 823 amino acids deduced from this renal HIF-1alpha cDNA had 99%, 96%, and 90% identity with rat, mouse, or human HIF-1alpha deposited in GenBank, respectively. The 3'-untranslated region of HIF-1alpha mRNA from the rat kidney contained seven AUUUA instability elements, five of which were found to be conserved among rat, mouse, and human HIF-1alpha. Northern blot analyses demonstrated a corticomedullary gradient of HIF-1alpha mRNA expression in the kidney, with the greatest abundance in the renal inner medulla. Western blot analyses also detected a higher HIF-1alpha protein level in the nuclear extracts from the renal medulla than the renal cortex. A classic loop diuretic, furosemide (10 mg/kg ip), markedly increased renal medullary Po(2) levels from 22.5 to 52.2 mmHg, which was accompanied by a significant reduction of HIF-1alpha transcripts in renal medullary tissue. In in vitro experiments, low Po(2), but not elevated osmolarity, was found to significantly increase HIF-1alpha mRNA in renal medullary interstitial cells and inner medullary collecting duct cells. These results indicate that HIF-1alpha is more abundantly expressed in the renal medulla compared with the renal cortex. Increased abundance of HIF-1alpha mRNA in the renal medulla may represent an adaptive response of renal medullary cells to low Po(2).

The present study was designed to test the hypothesis that hypoxia-inducible factor-1alpha (HIF-1alpha)-mediated transcriptional activation contributes to increased expression of heme oxygenase (HO) genes in renal medullary interstitial cells (RMICs). By Northern blot analysis, HO-1 mRNA expression was found to significantly increase in response to reduction of PO(2) in culture medium. However, HO-2 mRNA was not altered by hypoxia. This hypoxia-induced upregulation of HO-1 mRNA was significantly blocked by HIF-1alpha inhibition with ferrous ammonium sulfate. To further determine the role of HIF-1alpha in the activation of HO-1, the inducers of HIF-1alpha were used to address whether induction of HIF-1alpha stimulates HO-1 mRNA expression. Both desferrioxamine and CoCl(2) markedly increased HIF-1alpha mRNA and protein levels and resulted in the upregulation of HO-1 mRNA but not HO-2. Furthermore, inhibition of HIF-1alpha degradation by CBZ-LLL, an inhibitor of ubiquitin-proteasome, significantly increased HIF-1alpha protein and HO-1 mRNA but not HO-2 in these cells. Using cis-element oligodeoxynucleotide transfection to specifically decoy HIF-1alpha and block HIF-1alpha binding, increased mRNA expression of HO-1 in response to hypoxia and CoCl(2) was attenuated. In vitro nuclear run-on assays further confirmed that hypoxia and alterations of HIF-1alpha mRNA or protein levels significantly affected the formation of HO-1 mRNA. Taken together, our results indicate that HO-1, but not HO-2, is transcriptionally activated by hypoxia through HIF-1alpha-mediated mechanism in RMICs. This hypoxia-induced transcriptional activation may be one of the important mechanisms mediating increased expression of HO-1 in the renal medulla.

These results indicate that Hcys-induced alterations of ECM metabolism in mesangial cells are associated with enhanced NADH oxidase activity and that oxidative stress-stimulated up-regulation of TIMP-1 may play an important role in the deposition of collagen or ECM elements in the glomeruli during hHcys.

Introduction: 4-1BB (CD137, TNFRSF9) receptor agonists enhance cytotoxic T-cell and NK cell responses, including antibody (Ab)-dependent cellular cytotoxicity, and have shown antitumor activity in preclinical models. Utomilumab (Uto), a fully human IgG2 monoclonal Ab, binds to human 4-1BB with high affinity and specificity and activates 4-1BB while blocking binding to endogenous 4-1BB ligand. Initial findings from the dose-finding cohorts of this study were presented previously. We report here updated results and data from the expansion cohort in patients (pts) with rituximab (R)-refractory follicular lymphoma (FL). Methods:The dose-finding component of this phase I study evaluated Uto at doses ranging from 0.03 to 10 mg/kg in combination with R at the standard 375 mg/m 2 dose in pts with relapsed or refractory CD20 + non-Hodgkin's lymphoma (NHL), using a time-to-event continuousreassessment-method design. In the expansion cohort, pts with Rrefractory FL received Uto at the 1.2 mg/kg dose level. Pts received Uto on day 1, every 4 weeks up to 24 months and R from day 7, weekly for 4 weeks. The primary endpoint was dose-limiting toxicity (DLT) in the first 2 cycles, with safety, pharmacokinetics, and antitumor activity as secondary endpoints, and pharmacodynamics as an exploratory endpoint.Results: As of Nov 2016, 48 pts with CD20 + NHL including FL (n = 33), mantle cell (n = 6), diffuse large B cell (n = 3), marginal zone (n = 2), small lymphocytic (n = 2), and other (n = 2) lymphoma were treated with Uto combined with R. Approximately 50% of pts had received ≥3 prior anticancer therapies; 24/33 pts with FL had R-refractory disease. No DLTs were observed, and no pt discontinued treatment due to treatment-related adverse events (AEs). Of the 48 treated pts, 27% had grade ≥ 3 treatment-emergent AEs. The most common treatment-related AEs were fatigue (25%), infusion-related reaction (23%), and diarrhea (10%). Uto exposure appeared to increase with increasing doses. One (2%) of 43 anti-drug Ab (ADA) evaluable pts had treatmentinduced ADA/neutralizing Ab against Uto. The objective response rate (ORR) across all Uto dose levels tested was 23% (11/48). In all FL pts, the ORR was 27% (9/33) and 33% (8/24) in FL pts with R-refractory disease with 4 complete and 4 partial responses. In the expansion cohort of pts with R-refractory FL, the ORR was 44% (4/9). Pharmacodynamic effects of Uto, including increases in circulating CD8 + T cells and soluble 4-1BB, were observed at dose levels between 0.06 and 10 mg/kg. Enrollment into the expansion is ongoing. Conclusions:The combination of Uto with R showed a highly favorable tolerability profile with no DLTs observed and no substantial hematologic, hepatic, or immune-related toxicity reported. The preliminary evidence of clinical activity observed in R-refractory FL pts supports further evaluation of Uto plus R, especially in pts requiring treatment regimens with reduced toxicity.Keywords: B-cell lymphoma; non-Hodgkin lymphoma (NHL); rituximab. EXPRESSION OF LAG-3 DEFINES EXHAUS...