Shannon P. Hilchey

Increased rejection of primary tumors in mice lacking B cells : Inhibition of anti-tumor CTL and Th1 cytokine responses by B cells

We investigated the role of B cells in tumor immunity by studying immune responses of mice genetically lacking B cells to primary tumors. IgM−/− B cell‐deficient mice (BCDM) exhibited enhanced resistance to 3 histologically diverse syngeneic tumors as compared to the wild‐type (WT) mice. EL4 thymoma and MC38 colon carcinoma grew progressively in WT mice, but regressed spontaneously in BCDM whereas growth of B16 melanoma was slowed significantly in BCDM as compared to the WT mice. BCDM exhibited increased T cell infiltration of tumors, higher TH1 cytokine response and, in the case of MC38, a higher anti‐tumor CTL response. The increased tumor resistance of BCDM did not seem to result from intrinsic changes in their non‐B immunocytes because adoptive transfer of WT splenic B cells to BCDM abrogated tumor rejection and resulted in diminished anti‐tumor TH1 cytokine and CTL responses. Studies involving BCR‐transgenic mice indicated that B cells may inhibit anti‐tumor T cell responses by antigen‐nonspecific mechanisms since neither tumor‐specific antibodies nor cognate T:B interactions were necessary for inhibition of tumor immunity by B cells. IFN‐γ secretion in splenocyte:tumor co‐cultures of tumor‐challenged BCDM was inhibited by WT but not CD40−/− B cells indicating that B cells may inhibit anti‐tumor TH1 cytokine responses in a CD40‐dependent manner. Adoptive transfer of CD40−/− B cells into BCDM resulted in restored growth of MC38 suggesting additional factors other than CD40 are involved in dampening anti‐tumor responses. The effects of B cells on anti‐tumor response warrant further study.

Follicular Lymphoma Intratumoral CD4+CD25+GITR+ Regulatory T Cells Potently Suppress CD3/CD28-Costimulated Autologous and Allogeneic CD8+CD25− and CD4+CD25− T Cells

Regulatory T cells (TR) play a critical role in the inhibition of self-reactive immune responses and as such have been implicated in the suppression of tumor-reactive effector T cells. In this study, we demonstrate that follicular lymphoma (FL)-infiltrating CD8+ and CD4+ T cells are hyporesponsive to CD3/CD28 costimulation. We further identify a population of FL-infiltrating CD4+CD25+GITR+ TR that are significantly overrepresented within FL nodes (FLN) compared with that seen in normal (nonmalignant, nonlymphoid hyperplastic) or reactive (nonmalignant, lymphoid hyperplastic) nodes. These TR actively suppress both the proliferation of autologous nodal CD8+CD25− and CD4+CD25− T cells, as well as cytokine production (IFN-γ, TNF-α and IL-2), after CD3/CD28 costimulation. Removal of these cells in vitro by CD25+ magnetic bead depletion restores both the proliferation and cytokine production of the remaining T cells, demonstrating that FLN T cell hyporesponsiveness is reversible. In addition to suppressing autologous nodal T cells, these TR are also capable of suppressing the proliferation of allogeneic CD8+CD25− and CD4+CD25− T cells from normal lymph nodes as well as normal donor PBL, regardless of very robust stimulation of the target cells with plate-bound anti-CD3 and anti-CD28 Abs. The allogeneic suppression is not reciprocal, as equivalent numbers of CD25+FOXP3+ cells derived from either normal lymph nodes or PBL are not capable of suppressing allogeneic CD8+CD25− and CD4+CD25− T cells, suggesting that FLN TR are more suppressive than those derived from nonmalignant sources. Lastly, we demonstrate that inhibition of TGF-β signaling partially restores FLN T cell proliferation suggesting a mechanistic role for TGF-β in FLN TR-mediated suppression.

The mitochondrial ATP-dependent Lon protease: a novel target in lymphoma death mediated by the synthetic triterpenoid CDDO and its derivatives

Synthetic triterpenoids are multitarget compounds exhibiting promise as preventative and therapeutic agents for cancer. Their proposed mechanism of action is by forming Michael adducts with reactive nucleophilic groups on target proteins. Our previous work demonstrates that the 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivatives promote B-lymphoid cell apoptosis through a mitochondria-mediated pathway linked to mitochondrial protein aggregation. As one function of the Lon protease is to eliminate abnormal mitochondrial proteins, we hypothesized that CDDO-induced protein aggregation and lymphoma apoptosis occur by inactivating this enzyme. Here, we show that CDDO and its derivatives directly and selectively inhibit Lon. CDDO blocks Lon-mediated proteolysis in biochemical and cellular assays, but does not inhibit the 20S proteasome. Furthermore, a biotinylated-CDDO conjugate modifies mitochondrial Lon. A striking common phenotype of CDDO-treated lymphoma cells and Lon-knockdown cells is the accumulation of electron-dense aggregates within mitochondria. We also show that Lon protein levels are substantially elevated in malignant lymphoma cells, compared with resting or activated B cells. Finally, we demonstrate that Lon knockdown leads to lymphoma cell death. Together, these findings suggest that Lon inhibition plays a contributory role in CDDO-induced lymphoma cell death, and support the concept that mitochondrial Lon is a novel anticancer drug target.

Human Follicular Lymphoma CD39+-Infiltrating T Cells Contribute to Adenosine-Mediated T Cell Hyporesponsiveness

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.

Modulation of Cell Surface Protein Free Thiols: A Potential Novel Mechanism of Action of the Sesquiterpene Lactone Parthenolide

Background There has been much interest in targeting intracellular redox pathways as a therapeutic approach for cancer. Given recent data to suggest that the redox status of extracellular protein thiol groups (i.e. exofacial thiols) effects cell behavior, we hypothesized that redox active anti-cancer agents would modulate exofacial protein thiols. Methodology/Principal Findings To test this hypothesis, we used the sesquiterpene lactone parthenolide, a known anti-cancer agent. Using flow cytometry, and western blotting to label free thiols with Alexa Fluor 633 C5 maleimide dye and N-(biotinoyl)-N-(iodoacetyl) ethylendiamine (BIAM), respectively, we show that parthenolide decreases the level of free exofacial thiols on Granta mantle lymphoma cells. In addition, we used immuno-precipitation techniques to identify the central redox regulator thioredoxin, as one of the surface protein thiol targets modified by parthenolide. To examine the functional role of parthenolide induced surface protein thiol modification, we pretreated Granta cells with cell impermeable glutathione (GSH), prior to exposure to parthenolide, and showed that GSH pretreatment; (a) inhibited the interaction of parthenolide with exofacial thiols; (b) inhibited parthenolide mediated activation of JNK and inhibition of NFκB, two well established mechanisms of parthenolide activity and; (c) blocked the cytotoxic activity of parthenolide. That GSH had no effect on the parthenolide induced generation of intracellular reactive oxygen species supports the fact that GSH had no effect on intracellular redox. Together these data support the likelihood that GSH inhibits the effect of parthenolide on JNK, NFκB and cell death through its direct inhibition of parthenolides modulation of exofacial thiols. Conclusions/Significance Based on these data, we postulate that one component of parthenolides anti-lymphoma activity derives from its ability to modify the redox state of critical exofacial thiols. Further, we propose that cancer cell exofacial thiols may be important and novel targets for therapy.

Use of CFSE to Monitor Ex Vivo Regulatory T-cell Suppression of CD4+ and CD8+ T-cell Proliferation within Unseparated Mononuclear Cells from Malignant and Non-Malignant Human Lymph Node Biopsies

Regulatory T-cells (Tregs) play a critical role in the inhibition of self-reactive immune responses and as such have been implicated in the suppression of anti-tumor immunity. A clearer understanding of the mechanisms by which Tregs suppress effector T-cell responses within the context of anti-tumor immunity may lead to more effective treatments. The study of Tregs, particularly in the context of ongoing active immune responses, has been challenging due to the lack of surface molecules truly unique to these cells. Several surface markers have been shown to be constitutively expressed by Tregs, such as high levels of CD25, GITR and CTLA-4, and thus have been useful for their study. However, the heterogeneity of surface marker expression still makes identifying Tregs ex vivo challenging. As such, the only means available, currently, to accurately identify Tregs ex vivo is through functional suppression assays. Tregs have been shown to inhibit a variety of cellular functions including T-cell proliferation and as such, in vitro inhibition of proliferation is routinely used as a measure of Treg-mediated suppression. Several assays currently exist to assay cellular proliferation, including [3H]thymidine incorporation and CFSE dilution. However, a limitation of using [3H]thymidine is the difficulty differentiating between proliferation of the target cells and that of the Tregs themselves. Due to the ability to differentiate by flow cytometric analysis between labeled and unlabelled cells using CFSE, in contrast to [3H]thymidine, it is possible to analyze the proliferation of labeled target cells separate from unlabeled Tregs in co-culture experiments. In addition, the use of multi-color flow cytometry allows for the analysis of different T-cell subsets simultaneously without the necessity to separate these cells. Thus, CFSE has several advantages to [3H]thymidine for analysis of cellular proliferation. Herein we describe our work utilizing CFSE labeling to assess, (1) proliferative responses of CD4+ and CD8+ T-cells in unseparated single cell suspensions from human lymph nodes and, (2) the ability of tumor infiltrating suppressive populations, including Tregs, isolated from neoplastic lymph nodes to suppress in vitro proliferation of allogeneic CD4+ and CD8+ T-cells isolated from peripheral blood of healthy donors.

Follicular lymphoma tumor-infiltrating T-helper (T(H)) cells have the same polyfunctional potential as normal nodal T(H) cells despite skewed differentiation.

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 (T(H)) compared with reactive and normal lymph node (NLN) T(H) cells. Relative to NLNs, FL cells have decreased proportions of naive and central memory but increased proportions of effector memory T(H) cells. We further show differences in the distribution and anatomical localization of CXCR5(+) T(H) 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 T(H)2 and T(H)17 cells is higher in FL cells than in reactive lymph nodes or NLNs. However, the proportion of T(H)1 and polyfunctional T(H) cells (producing multiple cytokines simultaneously) is similar in FL cells and NLNs. These data suggest that, although T(H)-cell differentiation in FL is skewed compared with NLNs, FL T(H) cells should have the same intrinsic ability to elicit antitumor effector responses as NLN T(H) cells when tumor suppressive mechanisms are attenuated.

Dual targeting of the thioredoxin and glutathione antioxidant systems in malignant B cells: a novel synergistic therapeutic approach.

B-cell malignancies are a common type of cancer. One approach to cancer therapy is to either increase oxidative stress or inhibit the stress response systems on which cancer cells rely. In this study, we combined nontoxic concentrations of Auranofin (AUR), an inhibitor of the thioredoxin system, with nontoxic concentrations of buthionine-sulfoximine (BSO), a compound that reduces intracellular glutathione levels, and investigated the effect of this drug combination on multiple pathways critical for malignant B-cell survival. Auranofin interacted synergistically with BSO at low concentrations to trigger death in multiple malignant B-cell lines and primary mantle-cell lymphoma cells. Additionally, there was less toxicity toward normal B cells. Low AUR concentrations inhibited thioredoxin reductase (TrxR) activity, an effect significantly increased by BSO cotreatment. Overexpression of TrxR partially reversed AUR+BSO toxicity. Interestingly, the combination of AUR+BSO inhibited nuclear factor κB (NF-κB) signaling. Moreover, synergistic cell death induced by this regimen was attenuated in cells overexpressing NF-κB proteins, arguing for a functional role for NF-κB inhibition in AUR+BSO-mediated cell death. Together, these findings suggest that AUR+BSO synergistically induces malignant B-cell death, a process mediated by dual inhibition of TrxR and NF-κB, and such an approach warrants further investigation in B-cell malignancies.

DNA-based Loss of Specificity Mutations EFFECTS OF DNA SEQUENCE ON THE CONTACTED AND NON-CONTACTED BASE PREFERENCES OF BACTERIOPHAGE P22 REPRESSOR

Although the two central bases of the P22 operator are not contacted by the P22 repressor, changes in these bases alter the affinity of operator for repressor. Previous studies (Wu, L., and Koudelka, G. B. (1993) J. Biol. Chem. 268, 18975-18981) show that the structure of the P22 repressor-operator complex varies with central base sequence. Here we show that central base sequence composition affects the strength of two, and likely all, specific amino acid-base pair contacts between synthetic P22 operators and P22 repressor. However, altering a specific protein-DNA contact via a loss-of-contact mutation in repressor results in a loss of specificity at only one contacted position. Thus, only changing the sequence of non-contacted bases affects repressors global base specificity. The observed effects of ionic concentration on the affinities of various operators for repressor and the DNase I patterns of protein complexes with these binding sites indicate certain central base sequences facilitate optimal juxtaposition of repressor with its contacted bases, while others prevent it. The existence of different structural forms of the repressor-operator complexes explains how the relative energetic importance of specific amino acid-base pair edge contacts is modulated.

Recognition of nonconserved bases in the P22 operator by P22 repressor requires specific interactions between repressor and conserved bases.

The ability of P22 repressor protein to distinguish between the six naturally occurring operator binding sites is critically important in determining whether the bacteriophage chooses to grow lytically or lysogenically. We have shown that changes in the highly conserved bases at P22 operator positions 3, 5, 6, and 7 prevent specific binding of P22 repressor. Moreover, studies of mutant proteins identified the three repressor amino acids that directly contact these conserved bases. The pattern of operator sequence conservation permits these direct amino acid-base pair interactions to occur in all except one of the 12 operator half-sites in the phage chromosome. Therefore, repressor differential affinity for these sites cannot be due to these highly conserved base pair-amino acid interactions. Our binding studies show that the nonconserved bases at positions 2 and 4 also play an important role in determining the relative affinity of the naturally occurring P22 operators for P22 repressor. Our data indicate that the direct contacts between the three solvent-exposed amino acids and the conserved bases in the binding site lock these amino acids in place, forming a scaffold allowing the rest of the amino acids side chains to form weaker interactions with the nonconserved bases in the binding site.