Combination of Autophagy Selective Therapeutics With Doxil: An Assessment of Pathological Toxicity.

Background: Combination therapy of targeted drugs in cancer treatment is a field in constant flux, with research balancing side effects with efficacy. Efficacy from combination therapy is improved either through synthetic lethality or through prevention of recurrent clones. Previous research has shown (hydroxy-)chloroquine is insufficient to disrupt autophagy in tumors. Hence, either combinations or novel autophagy agents are desired. In vivo studies of ovarian cancer have revealed that chloroquine can be combined with up to four other autophagy drugs to suppress ovarian cancer growth. While cancer efficacy is now established for the autophagy drug combination, it is unclear what toxicities may require monitoring in human trials. Additive toxicity with chemotherapy is also unknown. Methods: To address toxicity in more depth than previous weight-monitoring studies, biochemical and histopathology studies were performed. Mouse groups were treated with autophagy drugs for 2 weeks, with or without the chemotherapy Doxil. After the last dose, mice were processed for blood biochemistry, white blood cell markers, and histopathology. Results: Data from a comprehensive blood biochemistry panel, flow cytometric measurements of blood cell markers, and histopathology are herein reported. While Doxil presented clear bone marrow and immunologic toxicity, autophagy drugs were overall less toxic and more variable in their presentation of potential toxicities. Only minor additive effects of autophagy drugs with Doxil were observed. Conclusion: Combinations of autophagy drugs may be considered for therapy in human oncology trials, with possible side effects to monitor informed by these murine pre-clinical data.

Activation of T cell checkpoint pathways during ß-cell antigen presentation by engineered dendritic cells promotes protection from type 1 diabetes.

Defective immune regulation has been recognized in type 1 diabetes (T1D). Immune regulatory T cell check-point receptors, which are generally upregulated on activated T cells, have been the molecules of attention as therapeutic targets for enhancing immune response in tumour therapy. Here, we show that pancreatic ß-cell antigen (BcAg) presentation by engineered tolerogenic dendritic cells (tDCs) that express CTLA4 selective ligand (B7.1wa) or a combination of CTLA4, PD1 and BTLA selective ligands (B7.1wa, PD-L1 and HVEM-CRD1 respectively; multiligand-DCs) causes an increase in regulatory cytokine and T cell (Treg) responses and suppression of the effector T cell function as compared with engineered control-DCs. Non-obese diabetic mice treated with BcAg-pulsed CTLA4-ligand-DCs and multiligand-DCs at pre-diabetic and early-hyperglycaemic stages showed significantly lower degree of insulitis, higher frequencies of insulin-positive islets, profound delay in and reversal of hyperglycaemia for a significant duration. Immune cells from the tDC-treated mice not only produced lower amounts of IFNγ and higher amounts of IL10 and TGFß1 upon BcAg challenge, but also failed to induce hyperglycaemia upon adoptive transfer. While both CTLA4-ligand-DCs and multiligand-DCs were effective in inducing tolerance, multiligand-DC treatment produced an overall higher suppressive effect on effector T cell function and disease outcome. These studies show that enhanced engagement of T cell checkpoint receptors during BcAg presentation can modulate T cell function and suppress autoimmunity and progression of the disease in T1D.

Gastrin producing syngeneic mesenchymal stem cells protect non-obese diabetic mice from type 1 diabetes.

Progressive destruction of pancreatic islet ß-cells by immune cells is a primary feature of type 1 diabetes (T1D) and therapies that can restore the functional ß-cell mass are needed to alleviate disease progression. Here, we report the use of mesenchymal stromal/stem cells (MSCs) for the production and delivery of Gastrin, a peptide hormone that is produced by intestinal cells and foetal islets and can increase ß-Cell mass, to promote protection from T1D. A single injection of syngeneic MSCs that were engineered to express Gastrin (Gastrin-MSCs) caused a significant delay in hyperglycaemia in non-obese diabetic (NOD) mice compared to engineered control-MSCs. Similar treatment of early-hyperglycaemic mice caused the restoration of euglycemia for a considerable duration, and these therapeutic effects were associated with the protection of, and/or higher frequencies of, insulin-producing islets and less severe insulitis. While the overall immune cell phenotype was not affected profoundly upon treatment using Gastrin-MSCs or upon in vitro culture, pancreatic lymph node cells from Gastrin-MSC treated mice, upon ex vivo challenge with self-antigen, showed a Th2 and Th17 bias, and diminished the diabetogenic property in NOD-Rag1 deficient mice suggesting a disease protective immune modulation under Gastrin-MSC treatment associated protection from hyperglycaemia. Overall, this study shows the potential of production and delivery of Gastrin in vivo, by MSCs, in protecting insulin-producing ß-cells and ameliorating the disease progression in T1D.

Centrosomal P4.1-associated protein (CPAP) positively regulates endocytic vesicular transport and lysosome targeting of EGFR.

Centrosomal P4.1-associated protein (CPAP) plays a critical role in restricting the centriole length in human cells. Here, we report a novel, positive regulatory influence for CPAP on endocytic vesicular transport (EVT) and lysosome targeting of internalized-cell surface receptor EGFR. We observed that higher CPAP levels cause an increase in the abundance of multi-vesicular body (MVB) and EGFR is detectable in CPAP-overexpression induced puncta. The surface and cellular levels of EGFR are higher under CPAP deficiency and lower under CPAP overexpression. While ligand-engagement induced internalization or routing of EGFR into early endosomes is not influenced by cellular levels of CPAP, we found that targeting of ligand-activated, internalized EGFR to lysosome is impacted by CPAP levels. Transport of ligand-bound EGFR from early endosome to late endosome/MVB and lysosome is diminished in CPAP-depleted cells. Moreover, CPAP depleted cells appear to show a diminished ability to form MVB structures upon EGFR activation. These observations suggest a positive regulatory effect of CPAP on EVT of ligand-bound EGFR-like cell surface receptors to MVB and lysosome. Overall, identification of a non-centriolar function of CPAP in endocytic trafficking provides new insights in understanding the non-canonical cellular functions of CPAP.

Loss of CPAP causes sustained EGFR signaling and epithelial-mesenchymal transition in oral cancer.

Higher epidermal growth factor receptor (EGFR) signaling can contribute to tumor metastasis and resistance to therapies in oral squamous cell carcinoma (OSCC). EGFR signaling can promote epithelial-mesenchymal transition (EMT) in OSCC. EMT is a process by which epithelial cells acquire invasive properties and it can contribute to tumor metastasis. Not only do the abnormal functions of microtubule and microtubule-organizing centers (MTOC) such as centrosomes lead to cancers, but also the malignant tissues are characterized by aberrant centriolar features and amplified centrosomes. Microtubule inhibition therapies increase the sensitivity to EGFR targeting drugs in various cancers. In this study, we show that the loss of expression of a microtubule/tubulin binding protein, centrosomal protein 4.1-associated protein (CPAP), which is critical for centriole biogenesis and normal functioning of the centrosome, caused an increase in the EGFR levels and its signaling and, enhanced the EMT features and invasiveness of OSCC cells. Further, depletion of CPAP enhanced the tumorigenicity of these cells in a xeno-transplant model. Importantly, CPAP loss-associated EMT features and invasiveness of multiple OSCC cells were attenuated upon depletion of EGFR in them. On the other hand, we found that CPAP protein levels were higher in EGF treated OSCC cells as well as in oral cancer tissues, suggesting that the frequently reported aberrant centriolar features of tumors are potentially a consequence, but not the cause, of tumor progression. Overall, our novel observations show that, in addition to its known indispensable role in centrosome biogenesis, CPAP also plays a vital role in suppressing tumorigenesis in OSCC by facilitating EGFR homeostasis.

Fungal ß-glucan, a Dectin-1 ligand, promotes protection from type 1 diabetes by inducing regulatory innate immune response.

ß-Glucans are naturally occurring polysaccharides in cereal grains, mushrooms, algae, or microbes, including bacteria, fungi, and yeast. Immune cells recognize these ß-glucans through a cell surface pathogen recognition receptor called Dectin-1. Studies using ß-glucans and other Dectin-1 binding components have demonstrated the potential of these agents in activating the immune cells for cancer treatment and controlling infections. In this study, we show that the ß-glucan from Saccharomyces cerevisiae induces the expression of immune regulatory cytokines (IL-10, TGF-ß1, and IL-2) and a tolerogenic enzyme (IDO) in bone marrow-derived dendritic cells as well as spleen cells. These properties can be exploited to modulate autoimmunity in the NOD mouse model of type 1 diabetes (T1D). Treatment of prediabetic NOD mice with low-dose ß-glucan resulted in a profound delay in hyperglycemia, and this protection was associated with increase in the frequencies of Foxp3(+), LAP(+), and GARP(+) T cells. Upon Ag presentation, ß-glucan-exposed dendritic cells induced a significant increase in Foxp3(+) and LAP(+) T cells in in vitro cultures. Furthermore, systemic coadministration of ß-glucan plus pancreatic ß cell Ag resulted in an enhanced protection of NOD mice from T1D as compared with treatment with ß-glucan alone. These observations demonstrate that the innate immune response induced by low-dose ß-glucan is regulatory in nature and can be exploited to modulate T cell response to ß cell Ag for inducing an effective protection from T1D.

Centrobin-centrosomal protein 4.1-associated protein (CPAP) interaction promotes CPAP localization to the centrioles during centriole duplication.

Centriole duplication is the process by which two new daughter centrioles are generated from the proximal end of preexisting mother centrioles. Accurate centriole duplication is important for many cellular and physiological events, including cell division and ciliogenesis. Centrosomal protein 4.1-associated protein (CPAP), centrosomal protein of 152 kDa (CEP152), and centrobin are known to be essential for centriole duplication. However, the precise mechanism by which they contribute to centriole duplication is not known. In this study, we show that centrobin interacts with CEP152 and CPAP, and the centrobin-CPAP interaction is critical for centriole duplication. Although depletion of centrobin from cells did not have an effect on the centriolar levels of CEP152, it caused the disappearance of CPAP from both the preexisting and newly formed centrioles. Moreover, exogenous expression of the CPAP-binding fragment of centrobin also caused the disappearance of CPAP from both the preexisting and newly synthesized centrioles, possibly in a dominant negative manner, thereby inhibiting centriole duplication and the PLK4 overexpression-mediated centrosome amplification. Interestingly, exogenous overexpression of CPAP in the centrobin-depleted cells did not restore CPAP localization to the centrioles. However, restoration of centrobin expression in the centrobin-depleted cells led to the reappearance of centriolar CPAP. Hence, we conclude that centrobin-CPAP interaction is critical for the recruitment of CPAP to procentrioles to promote the elongation of daughter centrioles and for the persistence of CPAP on preexisting mother centrioles. Our study indicates that regulation of CPAP levels on the centrioles by centrobin is critical for preserving the normal size, shape, and number of centrioles in the cell.

OX40L/Jagged1 cosignaling by GM-CSF-induced bone marrow-derived dendritic cells is required for the expansion of functional regulatory T cells.

Earlier, we had demonstrated that treatment with low dose of GM-CSF can prevent the development of experimental autoimmune thyroiditis (EAT), experimental autoimmune myasthenia gravis, and type 1 diabetes, and could also reverse ongoing EAT and experimental autoimmune myasthenia gravis. The protective effect was mediated through the induction of tolerogenic CD11C(+)CD8α(-) dendritic cells (DCs) and consequent expansion of Foxp3(+) regulatory T cells (Tregs). Subsequently, we showed that GM-CSF acted specifically on bone marrow precursors and facilitated their differentiation into tolerogenic dendritic cells (DCs; GM-CSF-induced bone marrow-derived DCs [GM-BMDCs]), which directed Treg expansion in a contact-dependent manner. This novel mechanism of Treg expansion was independent of TCR-mediated signaling but required exogenous IL-2 and cosignaling from DC-bound OX40L. In this study, we observed that OX40L-mediated signaling by GM-BMDCs, although necessary, was not sufficient for Treg expansion and required signaling by Jagged1. Concurrent signaling induced by OX40L and Jagged1 via OX40 and Notch3 receptors expressed on Tregs was essential for the Treg expansion with sustained FoxP3 expression. Adoptive transfer of only OX40L(+)Jagged1(+) BMDCs led to Treg expansion, increased production of IL-4 and IL-10, and suppression of EAT in the recipient mice. These results showed a critical role for OX40L- and Jagged1-induced cosignaling in GM-BMDC-induced Treg expansion.

IL-1ß promotes TGF-ß1 and IL-2 dependent Foxp3 expression in regulatory T cells.

Earlier, we have shown that GM-CSF-exposed CD8α- DCs that express low levels of pro-inflammatory cytokines IL-12 and IL-1ß can induce Foxp3+ Tregs leading to suppression of autoimmunity. Here, we examined the differential effects of IL-12 and IL-1ß on Foxp3 expression in T cells when activated in the presence and absence of DCs. Exogenous IL-12 abolished, but IL-1ß enhanced, the ability of GM-CSF-exposed tolerogenic DCs to promote Foxp3 expression. Pre-exposure of DCs to IL-1ß and IL-12 had only a modest effect on Foxp3- expressing T cells; however, T cells activated in the absence of DCs but in the presence of IL-1ß or IL-12 showed highly significant increase and decrease in Foxp3+ T cell frequencies respectively suggesting direct effects of these cytokines on T cells and a role for IL-1ß in promoting Foxp3 expression. Importantly, purified CD4+CD25+ cells showed a significantly higher ability to maintain Foxp3 expression when activated in the presence of IL-1ß. Further analyses showed that the ability of IL-1ß to maintain Foxp3 expression in CD25+ T cells was dependent on TGF-ß1 and IL-2 expression in Foxp3+Tregs and CD25- effectors T cells respectively. Exposure of CD4+CD25+ T cells to IL-1ß enhanced their ability to suppress effector T cell response in vitro and ongoing experimental autoimmune thyroidits in vivo. These results show that IL-1ß can help enhance/maintain Tregs, which may play an important role in maintaining peripheral tolerance during inflammation to prevent and/or suppress autoimmunity.

Modulation of dendritic cells using granulocyte-macrophage colony-stimulating factor (GM-CSF) delays type 1 diabetes by enhancing CD4+CD25+ regulatory T cell function.

Abnormalities in DC function are implicated in defective immune regulation that leads to type-1 diabetes (T1D) in NOD mice and humans. In this study, we used GM-CSF and Flt3-L to modulate DC function in NOD mice and observed the effects on T1D development. Treatment with either ligand at earlier stages of insulitis suppressed the development of T1D. Unlike Flt3-L, GM-CSF was more effective in suppressing T1D, even when administered at later stages of insulitis. In vitro studies and in vivo adoptive transfer experiments revealed that CD4+CD25+ T cells from GM-CSF-treated mice could suppress effector T cell response and T1D. This suppression is likely mediated through enhanced IL-10 and TGF-beta1 production. Adoptive transfer of GM-CSF exposed DCs to naive mice resulted in an expansion of Foxp3+ T cells and a significant delay in T1D onset. Our results indicate that GM-CSF acted primarily on DCs and caused an expansion of Foxp3+ Tregs which delayed the onset of T1D in NOD mice.

GM-CSF-induced CD11c+CD8a--dendritic cells facilitate Foxp3+ and IL-10+ regulatory T cell expansion resulting in suppression of autoimmune thyroiditis.

GM-CSF plays an essential role in the differentiation of dendritic cells (DCs). Our studies have shown that GM-CSF treatment can induce semi-mature DCs and CD4+CD25+ regulatory T cells (Tregs) and suppress ongoing autoimmunity in mouse models. In this study, we examined the differences in the potential of GM-CSF to exert tolerogenic function on CD8a+ and CD8a- sub-populations of DCs in vivo. We show that GM-CSF modulates CD8a-, but not CD8a+ DCs in vivo, by inhibiting the surface expression of activation markers MHC II and CD80 and production of inflammatory cytokines such as IL-12 and IL-1beta. Self-antigen [mouse thyroglobulin (mTg)] presentation by GM-CSF-exposed CD8a- DCs to T cells from mTg-primed mice induced a profound increase in the frequency of forkhead box P3 (FoxP3)-expressing T cells compared with antigen presentation by GM-CSF-exposed CD8a+ DCs and control CD8a+ and CD8a- DCs. This tolerogenic property of GM-CD8a- DCs was abrogated when IL-12 was added. GM-CSF-exposed CD8a- DCs could also induce secretion of significantly higher amounts of IL-10 by T cells from mTg-primed mice. Importantly, adoptive transfer of CD8a- DCs from GM-CSF-treated SCID mice, but not untreated mice, into wild-type CBA/J mice prevented the development of experimental autoimmune thyroiditis (EAT) in the recipient animals upon immunization with mTg. Collectively, our results show that GM-CSF renders CD8a- DCs tolerogenic, and these DCs induce Foxp3+ and IL-10+ Tregs.

Autonomic denervation of lymphoid organs leads to epigenetic immune atrophy in a mouse model of Krabbe disease.

Lysosomal beta-galactosylceramidase deficiency results in demyelination and inflammation in the nervous system causing the neurological Krabbe disease. In the Twitcher mouse model of this disease, we found that neurological symptoms parallel progressive and severe lymphopenia. Although lymphopoiesis is normal before disease onset, primary and secondary lymphoid organs progressively degenerate afterward. This occurs despite preserved erythropoiesis and leads to severe peripheral lymphopenia caused by reduced numbers of T cell precursors and mature lymphocytes. Hematopoietic cell replacement experiments support the existence of an epigenetic factor in mutant mice reconcilable with a progressive loss of autonomic axons that hampers thymic functionality. We propose that degeneration of autonomic nerves leads to the irreversible thymic atrophy and loss of immune-competence. Our study describes a new aspect of Krabbe disease, placing patients at risk of immune-related pathologies, and identifies a novel target for therapeutic interventions.

Enhanced engagement of CTLA-4 induces antigen-specific CD4+CD25+Foxp3+ and CD4+CD25- TGF-beta 1+ adaptive regulatory T cells.

CTLA-4 is a critical negative regulator of T cell response and is instrumental in maintaining immunological tolerance. In this article, we report that enhanced selective engagement of CTLA-4 on T cells by Ag-presenting dendritic cells resulted in the induction of Ag-specific CD4(+)CD25(+)Foxp3(+) and CD4(+)CD25(-)TGF-beta1(+) adaptive Tregs. These cells were CD62L(low) and hyporesponsive to stimulation with cognate Ag but demonstrated a superior ability to suppress Ag-specific effector T cell response compared with their CD62L(high) counterparts. Importantly, treatment of mice with autoimmune thyroiditis using mouse thyroglobulin (mTg)-pulsed anti-CTLA-4 agonistic Ab-coated DCs, which results in a dominant engagement of CTLA-4 upon self-Ag presentation, not only suppressed thyroiditis but also prevented reemergence of the disease upon rechallenge with mTg. Further, the disease suppression was associated with significantly reduced mTg-specific T cell and Ab responses. Collectively, our results showed an important role for selective CTLA-4 signaling in the induction of adaptive Tregs and suggested that approaches that allow dominant CTLA-4 engagement concomitant with Ag-specific TCR ligation can be used for targeted therapy.

Bone marrow is a preferential homing site for autoreactive T-cells in type 1 diabetes.

OBJECTIVE: The pancreatic microenvironment is considered to be the primary location of autoreactive T-cells in type 1 diabetes. Diabetogenic T-cells have also been detected in the spleens of NOD mice. However, it is not known whether bone marrow also contains T-cells specific for self-antigens in hosts with autoimmunity. In this study, we investigated whether autoreactive diabetogenic T-cells are present in the bone marrow of NOD mice. RESEARCH DESIGN AND METHODS: Bone marrow and splenic T-cells of female NOD mice were purified and tested for their cytokine secretion and proliferation in response to stimulation with immunodominant peptides of pancreatic beta-cells. The diabetogenic nature and homing properties of purified bone marrow T-cells were compared with those of splenic T-cells in NOD-Scid and wild-type mice. RESULTS: The bone marrow T-cells from both hyperglycemic and young euglycemic mice demonstrated profoundly higher proliferation and cytokine production in response to stimulation with beta-cell antigens than T-cells from spleen. Bone marrow T-cells showed rapid expansion and aggressive infiltration into pancreatic islets in NOD-Scid mice and induced hyperglycemia earlier than splenic T-cells. Adoptive transfer of bone marrow T-cells resulted in their trafficking predominantly to bone marrow and pancreatic lymph nodes. CONCLUSIONS: Our study demonstrates that a large number of diabetogenic T-cells are present in the bone marrow of female NOD mice and that these autoreactive T-cells can be detected long before clinical onset of the disease.

Suppression of experimental autoimmune myasthenia gravis by granulocyte-macrophage colony-stimulating factor is associated with an expansion of FoxP3+ regulatory T cells.

Dendritic cells (DCs) have the potential to activate or tolerize T cells in an Ag-specific manner. Although the precise mechanism that determines whether DCs exhibit tolerogenic or immunogenic functions has not been precisely elucidated, growing evidence suggests that DC function is largely dependent on differentiation status, which can be manipulated using various growth factors. In this study, we investigated the effects of mobilization of specific DC subsets-using GM-CSF and fms-like tyrosine kinase receptor 3-ligand (Flt3-L)-on the susceptibility to induction of experimental autoimmune myasthenia gravis (EAMG). We administered GM-CSF or Flt3-L to C57BL/6 mice before immunization with acetylcholine receptor (AChR) and observed the effect on the frequency and severity of EAMG development. Compared with AChR-immunized controls, mice treated with Flt3-L before immunization developed EAMG at an accelerated pace initially, but disease frequency and severity was comparable at the end of the observation period. In contrast, GM-CSF administered before immunization exerted a sustained suppressive effect against the induction of EAMG. This suppression was associated with lowered serum autoantibody levels, reduced T cell proliferative responses to AChR, and an expansion in the population of FoxP3+ regulatory T cells. These results highlight the potential of manipulating DCs to expand regulatory T cells for the control of autoimmune diseases such as MG.

Members of adenovirus species B utilize CD80 and CD86 as cellular attachment receptors.

Alternate serotypes of adenovirus (Ad), including Ads of species B, are being explored to circumvent the disadvantages of Ad serotype 5 gene delivery vectors. Whereas the majority of human Ads utilize the Coxsackievirus and adenovirus receptor (CAR), none of the Ad species B use CAR. Ad species B is further divided into two subspecies, B1 and B2, and utilizes at least two classes of receptors: common Ad species B receptors and B2 specific receptors. CD46 has been implicated as a B2-specific receptor. Ad serotype 3 (Ad3), a member of B1, utilizes CD80 and CD86 as cellular attachment receptors. The receptor-interacting Ad fiber-knob domain is highly homologous among species B Ads. We hypothesized that other members of Ad species B may utilize CD80 and CD86 as cellular attachment receptors. All tested species B members showed specific binding to cells expressing CD80 and CD86, and the Ad fiber-knob domain from both B1 and B2 Ad efficiently blocked CD80- and CD86-mediated infection of Ad3 vectors. Members of both B1 and B2 demonstrated CD80- and CD86-specific infection of CHO cells expressing CD80 and CD86. Therefore, all of the members of Ad species B utilize CD80 and CD86 for infection of cells.

IL-10-producing CD4+CD25+ regulatory T cells play a critical role in granulocyte-macrophage colony-stimulating factor-induced suppression of experimental autoimmune thyroiditis.

Our earlier study showed that GM-CSF has the potential not only to prevent, but also to suppress, experimental autoimmune thyroiditis (EAT). GM-CSF-induced EAT suppression in mice was accompanied by an increase in the frequency of CD4(+)CD25(+) regulatory T cells that could suppress mouse thyroglobulin (mTg)-specific T cell responses in vitro, but the underlying mechanism of this suppression was not elucidated. In this study we show that GM-CSF can induce dendritic cells (DCs) with a semimature phenotype, an important characteristic of DCs, which are known to play a critical role in the induction and maintenance of regulatory T cells. Adoptive transfer of CD4(+)CD25(+) T cells from GM-CSF-treated and mTg-primed donors into untreated, but mTg-primed, recipients resulted in decreased mTg-specific T cell responses. Furthermore, lymphocytes obtained from these donors and recipients after adoptive transfer produced significantly higher levels of IL-10 compared with mTg-primed, untreated, control mice. Administration of anti-IL-10R Ab into GM-CSF-treated mice abrogated GM-CSF-induced suppression of EAT, as indicated by increased mTg-specific T cell responses, thyroid lymphocyte infiltration, and follicular destruction. Interestingly, in vivo blockade of IL-10R did not affect GM-CSF-induced expansion of CD4(+)CD25(+) T cells. However, IL-10-induced immunosuppression was due to its direct effects on mTg-specific effector T cells. Taken together, these results indicated that IL-10, produced by CD4(+)CD25(+) T cells that were probably induced by semimature DCs, is essential for disease suppression in GM-CSF-treated mice.

Targeted CTLA-4 engagement induces CD4+CD25+CTLA-4high T regulatory cells with target (allo)antigen specificity.

CTLA-4 (CD152) is actively involved in down-regulating T cell activation and maintaining lymphocyte homeostasis. Our earlier studies showed that targeted engagement of CTLA-4 can down-modulate T cell response and suppress allo- and autoimmune responses. In this study, we report that targeted CTLA-4 engagement can induce immune tolerance to a specific target through selective induction of an Ag-specific CD4(+)CD25(+)CTLA-4(high) regulatory T cell (Treg cell) population. Allogeneic cells coated with anti-CTLA-4 Ab induced immune hyporesponsiveness through suppression of proinflammatory cytokines IFN-gamma and IL-2, and up-regulation of the regulatory cytokines IL-10, TGF-beta1, and IL-4, presumably through the engagement of CTLA-4 on activated T cells. Although rechallenge with alloantigen failed to break the unresponsiveness, a transient recovery from tolerance was observed in the presence of high concentrations of exogenous IL-2, saturating concentrations of neutralizing anti-TGF-beta1 and anti-IL-10 Abs, and blocking anti-CTLA-4 Ab, and upon depletion of CD4(+)CD25(+) Treg cells. The CD4(+)CD25(+)CTLA-4(high) Treg cells from tolerant mice suppressed the effector function of CD25(-) T cells from Ag-primed mice. Adoptive transfer of these Treg cells into Ag-primed mice resulted in a significantly reduced alloantigen-specific response. Further characterization demonstrated that the Treg cells with memory phenotype (CD62L(-)) were more potent in suppressing the alloantigen-specific T cell response. These results strongly support that the targeted engagement of CTLA-4 has therapeutic potential for the prevention of transplant rejection.

Adenovirus serotype 3 utilizes CD80 (B7.1) and CD86 (B7.2) as cellular attachment receptors.

Most viruses exploit a variety of host cellular proteins as primary cellular attachment receptors in the context of successful execution of infection. Furthermore, many viral agents have evolved precise mechanisms to subvert host immune recognition to achieve persistence. Herein we present data indicating that adenovirus (Ad) serotype 3 utilizes CD80 (B7.1) and CD86 (B7.2) as cellular attachment receptors. CD80 and CD86 are co-stimulatory molecules that are present on mature dendritic cells and B lymphocytes and are involved in stimulating T-lymphocyte activation. To our knowledge, this is one of the first demonstrations of a virus utilizing immunologic accessory molecules as a primary means of cellular entry. This finding suggests a mechanism whereby viral exploitation of these proteins as receptors may achieve both goals of cellular entry and evading the immune system.

Bacterial cupredoxin azurin as an inducer of apoptosis and regression in human breast cancer.

Azurin, a copper-containing redox protein released by the pathogenic bacterium Pseudomonas aeruginosa, is highly cytotoxic to the human breast cancer cell line MCF-7, but is less cytotoxic toward p53-negative (MDA-MB-157) or nonfunctional p53 cell lines like MDD2 and MDA-MB-231. The purpose of this study was to investigate the underlying mechanism of the action of bacterial cupredoxin azurin in the regression of breast cancer and its potential chemotherapeutic efficacy. Azurin enters into the cytosol of MCF-7 cells and travels to the nucleus, enhancing the intracellular levels of p53 and Bax, thereby triggering the release of mitochondrial cytochrome c into the cytosol. This process activates the caspase cascade (including caspase-9 and caspase-7), thereby initiating the apoptotic process. Our results indicate that azurin-induced cell death stimuli are amplified in the presence of p53. In vivo injection of azurin in immunodeficient mice harboring xenografted human breast cancer cells in the mammary fat pad leads to statistically significant regression (85%, P = 0.0179, Kruskal-Wallis Test) of the tumor. In conclusion, azurin blocks breast cancer cell proliferation and induces apoptosis through the mitochondrial pathway both in vitro and in vivo, thereby suggesting a potential chemotherapeutic application of this bacterial cupredoxin for the treatment of breast cancer.