Partha Pratim Manna

CD47 Mediates Killing of Breast Tumor Cells via Gi-Dependent Inhibition of Protein Kinase A

Thrombospondins (TSPs) have been implicated as antitumor and antimetastasis factors in breast cancer. Although this effect has been attributed to the antiangiogenic activity of TSPs, recent observations suggest other mechanisms may be at work. The TSP receptor CD47 (integrin-associated protein) has recently been reported to mediate a novel form of apoptosis. Here, we have studied the response of breast cancer cells to CD47 ligands TSP-1, the CD47 agonist peptide 4N1K derived from TSP-1, and the anti-CD47 monoclonal antibody 1F7. All of these ligands killed four different breast cancer cell lines. This CD47-mediated cell death did not require active caspases or Bcl-2 degradation and did not cause DNA laddering or cytochrome c release. Pertussis toxin (PTX) prevented CD47-mediated death, indicating the involvement of Giα. 4N1K dramatically reduced intracellular cAMP levels, an effect reversed with PTX. Forskolin, 8-bromo cAMP, and isobutylmethylxanthine (IBMX) all prevented CD47-mediated apoptosis, indicating the involvement of cAMP. H89 and protein kinase A (PKA) inhibitor peptide prevented rescue of breast cancer cells by PTX, 8-Br-cAMP, and forskolin, suggesting that the effects of cAMP are mediated via PKA-dependent phosphorylation events. Epidermal growth factor also inhibited CD47-induced apoptosis via a PKC-dependent but ERK-independent pathway. Thus, CD47-mediated killing of breast cancer cells occurs by a novel pathway involving regulation of cAMP levels by heterotrimeric Gi with subsequent effects mediated by PKA.

The Mechanism of CD47-Dependent Killing of T Cells: Heterotrimeric Gi-Dependent Inhibition of Protein Kinase A

CD47 has been implicated in both positive and negative regulation of T cells as well as in T cell death. To clarify the role of CD47 in T cell function, we have studied the mechanism of T cell death in response to CD47 ligands, including mAb 1F7, thrombospondin-1, and a CD47 agonist peptide derived from it. CD47−/− Jurkat T cells (JINB8) were resistant to killing by all three ligands, indicating the essential role of CD47. Primary human T cells were also killed by CD47 ligands, but only after activation with anti-CD3. CD47-mediated cell death occurred without active caspases, DNA fragmentation, or Bcl-2 degradation. Pretreatment of Jurkat and primary T cells with pertussis toxin (PTX) prevented CD47-mediated death, indicating the involvement of Giα. Pretreatment of T cells with 8-bromo cAMP, forskolin, or 3-isobutyl-1-methylxanthine prevented the CD47-mediated apoptosis, and 1F7 dramatically reduced intracellular cAMP levels, an effect reversed with PTX. H89 and protein kinase A (PKA) inhibitor peptide, a specific PKA inhibitor, prevented rescue of T cells by PTX, 8-bromo cAMP, and forskolin, indicating a direct role for one or more PKA substrates. Thus, CD47-mediated killing of activated T cells occurs by a novel pathway involving regulation of cAMP levels by heterotrimeric Giα with subsequent effects mediated by PKA.

Identification of HLA-A3-restricted CD8+ T cell epitopes derived from mammaglobin-A, a tumor-associated antigen of human breast cancer.

Mammaglobin‐A is highly overexpressed in breast cancer cell lines and primary breast tumors. This pattern of expression is restricted to mammary epithelium and metastatic breast tumors. Thus, mammaglobin‐A‐specific T cell immune responses may provide an important approach for the design of breast cancer‐specific immunotherapy. The purpose of our study was to define the T cell‐mediated immune response to mammaglobin‐A. We determined that the frequency of mammaglobin‐A‐reactive CD8+ and CD4+ T cells in breast cancer patients is significantly higher than that observed in healthy female controls using limiting dilution analyses (p = 0.026 and p = 0.02, respectively). We identified 8 mammaglobin‐A‐derived 9‐mer peptides with the highest binding affinity for the HLA‐A3 molecule (Mam‐A3.1–8) using a computer‐assisted analysis of the mammaglobin‐A protein sequence. Subsequently, we determined that CD8+ T cells from breast cancer patients reacted to peptides Mam‐A3.1 (23–31, PLLENVISK), Mam‐A3.3 (2–10, KLLMVLMLA), Mam‐A3.4 (55–63, TTNAIDELK) and Mam‐A3.8 (58–66, AIDELKECF) using an IFN‐γ enzyme‐linked immunospot assay. A CD8+ T cell line generated in vitro against HLA‐A*0301‐transfected TAP‐deficient T2 cells loaded with these peptides showed significant cytotoxic activity against the Mam‐A3.1 peptide. This CD8+ T cell line showed a significant HLA‐A3‐restricted cytotoxic activity against mammaglobin‐A‐positive but not mammaglobin‐A‐negative breast cancer cells. In summary, our study identified four HLA‐A3‐restricted mammaglobin‐A‐derived epitopes naturally expressed by breast cancer cells, indicating the immunotherapeutic potential of this novel antigen for the treatment and prevention of breast cancer.

Deficit of CD47 Results in a Defect of Marginal Zone Dendritic Cells, Blunted Immune Response to Particulate Antigen and Impairment of Skin Dendritic Cell Migration

CD47 is a ubiquitously expressed cell surface glycoprotein that associates with integrins and regulates chemotaxis, migration, and activation of leukocytes. CD47 is also a ligand for signal regulatory protein α, a cell surface receptor expressed on monocytes, macrophages, granulocytes, and dendritic cell (DC) subsets that regulates cell activation, adhesion, and migration. Although the function of CD47 in macrophages and granulocytes has been studied in detail, little is known about the role of CD47 in DC biology in vivo. In this study we demonstrate that CD47−/− mice exhibit a selective reduction of splenic CD11chighCD11bhighCD8α−CD4+ DCs. These DCs correspond to marginal zone DCs and express signal regulatory protein α, possibly explaining their selective deficiency in CD47−/− mice. Deficiency of marginal zone DCs resulted in impairment of IgG responses to corpusculate T cell-independent Ags. Although epidermal DCs were present in normal numbers in CD47−/− mice, their migration to draining lymph nodes in response to contact sensitization was impaired, while their maturation was intact. In vitro, CD47−/− mature DCs showed normal CCR7 expression but impaired migration to CCL-19, whereas immature DC response to CCL-5 was only slightly impaired. These results demonstrate a fundamental role of CD47 in DC migration in vivo and in vitro and in the function of marginal zone DCs.

CD47 Augments Fas/CD95-mediated Apoptosis

Fas (CD95) mediates apoptosis of many cell types, but the susceptibility of cells to killing by Fas ligand and anti-Fas antibodies is highly variable. Jurkat T cells lacking CD47 (integrin-associated protein) are relatively resistant to Fas-mediated death but are efficiently killed by Fas ligand or anti-Fas IgM (CH11) upon expression of CD47. Lack of CD47 impairs events downstream of Fas activation including caspase activation, poly-(ADP-ribose) polymerase cleavage, cytochrome c release from mitochondria, loss of mitochondrial membrane potential, and DNA cleavage. Neither CD47 signaling nor raft association of CD47 is required to enable Fas apoptosis. CH11 induces association of Fas and CD47. Primary T cells from CD47-null mice are also protected from Fas-mediated killing relative to wild type T cells. Thus CD47 associates with Fas upon its activation and augments Fas-mediated apoptosis.

Human dendritic cell mediated cytotoxicity against breast carcinoma cells in vitro

Dendritic cells (DC) are an important subset of antigen‐presenting cells characterized by their potent capacity to activate immunologically naïve T cells. However, their role in effector function in tumor resistance is less well characterized. We report here that activated human peripheral blood DC acquire a potent antitumor effect against breast cancer cell lines in vitro, leading to growth inhibition and apoptosis of the tumor cell. The antitumor effect of DC was augmented by proinflammatory stimuli induced by lipopolysaccharide (LPS) treatment. Tumor necrosis factor α (TNF‐α) produced after DC activation was responsible for the antitumor activity of DC. Interferon‐γ, interleukin‐15, or LPS treatment of DC markedly augmented the effector function of DC against most of the breast cells, indicating heterogeneity of the tumor and its susceptibility to cytokine‐mediated damage. Treatment of LPS‐activated DC or cell‐free supernatant with anti‐human TNF‐α significantly reduces the antitumor effect against the tumor cells tested. These results suggest that in addition to their predominant role as immune regulatory cells, DC could serve as innate effector cells in tumor immunity.

Targeted delivery of doxorubicin-loaded poly (ε-caprolactone)-b-poly (N-vinylpyrrolidone) micelles enhances antitumor effect in lymphoma.

Background The present study was motivated by the need to design a safe nano-carrier for the delivery of doxorubicin which could be tolerant to normal cells. PCL63-b-PNVP90 was loaded with doxorubicin (6 mg/ml), and with 49.8% drug loading efficiency; it offers a unique platform providing selective immune responses against lymphoma. Methods In this study, we have used micelles of amphiphilic PCL63-b-PNVP90 block copolymer as nano-carrier for controlled release of doxorubicin (DOX). DOX is physically entrapped and stabilized in the hydrophobic cores of the micelles and biological roles of these micelles were evaluated in lymphoma. Results DOX loaded PCL63-b-PNVP90 block copolymer micelles (DOX-PCL63-b-PNVP90) shows enhanced growth inhibition and cytotoxicity against human (K-562, JE6.1 and Raji) and mice lymphoma cells (Daltons lymphoma, DL). DOX-PCL63-b-PNVP90 demonstrates higher levels of tumoricidal effect against DOX-resistant tumor cells compared to free DOX. DOX-PCL63-b-PNVP90 demonstrated effective drug loading and a pH-responsive drug release character besides exhibiting sustained drug release performance in in-vitro and intracellular drug release experiments. Conclusion Unlike free DOX, DOX-PCL63-b-PNVP90 does not show cytotoxicity against normal cells. DOX-PCL63-b-PNVP90 prolonged the survival of tumor (DL) bearing mice by enhancing the apoptosis of the tumor cells in targeted organs like liver and spleen.

Indirect Recognition of Porcine Swine Leukocyte Ag Class I Molecules Expressed on Islets by Human CD4+ T Lymphocytes

Xenotransplantation of porcine islets is considered a viable alternative treatment for type 1 diabetes mellitus. Therefore, we characterized human PBL responding to porcine islets both in vitro by coculture and in vivo using SCID mice reconstituted with human PBLs (HuPBL-SCID) and transplanted with porcine islets. T cell lines generated in vitro and graft-infiltrating T cells obtained from HuPBL-SCID mice were CD4+-proliferated specifically to porcine islets cultured with autologous APC. This proliferation was abrogated by an anti-human class II Ab. These T cell lines also proliferated to purified swine leukocyte Ag (SLA) class I molecules in the presence of self-APC, indicating that the primary xenoantigens recognized are peptides derived from SLA. This CD4+ T cell line lysed porcine islets but not splenocytes. CD4+ T cell clones with Th0, Th1, and Th2 cytokine profiles were isolated. The Th0 and Th1 clones lysed porcine islets, whereas the Th2 clone that secreted a large amount of IL-4 was not lytic. These results demonstrate that human T cells responding to porcine islets are primarily CD4+ and recognize porcine xenoantigens by the indirect Ag pathway presentation. These activated T cells produce cytokines that lyse islets. Furthermore, we demonstrate that the major porcine xenoantigens recognized are SLA class I molecules.

Differentiation and functional maturation of human CD14(+) adherent peripheral blood monocytes by xenogeneic endothelial cells: up-regulation of costimulation, cytokine generation, and toll-like receptors.

Background. Dendritic cells (DCs) can be generated from peripheral blood mononuclear cells (PBMC) after stimulation with exogenous granulocyte-macrophage colony stimulating factor and interleukin (IL)-4. Further, extravasation of monocytes through human endothelial cells can also cause differentiation, maturation, and expression of DC-specific phenotype. However, it is unclear whether human DCs can be generated from monocytes under the influence of xenogeneic endothelial cells in the absence of exogenous cytokines. We therefore analyzed and compared the effect of human and porcine endothelial cells on the differentiation of human monocytes into DC. Methods. Adherent peripheral blood CD14+ monocytes were cultured in the presence of different cytokine combinations, human or porcine endothelial cells, and smooth muscle cells, and analyzed for DC-specific antigen expression, antigen-presenting capacity, cytokine and chemokine generation, and expression of Toll-like receptors by flow cytometry and reverse transcription polymerase chain reaction. Results. Human monocytes express a DC-specific surface phenotype and efficiently present allo- and xenoantigens to allogeneic T cells after co-culturing with allogeneic and xenogeneic endothelial cells, respectively. Differentiation of monocytes under different stimulating conditions is also accompanied by the up-regulation of costimulatory molecules (CD40, CD80, CD86), adhesion molecules (CD54), human leukocyte antigen (HLA)-DR, synthesis of cytokines tumor necrosis factor-&agr;, IL-12p70, IL-10, and differential expression of message for Toll-like receptors. Conclusions. Porcine aortic endothelial cells can provide immunostimulatory signals to human peripheral blood adherent monocytes similar to allogeneic endothelial cells through the participation of innate immune mechanisms.

IL-15 activated human peripheral blood dendritic cell kill allogeneic and xenogeneic endothelial cells via apoptosis.

IL-15 is a pleotropic cytokine, which plays an important role in natural killer (NK) cell activity, T cell proliferation, and T cell cytotoxic activity. Dendritic cells (DCs) are the major antigen presenting cells in the immune system and presumed to play an important role in immune recognition of allo and xenotransplantation. We showed that IL-15 activated human peripheral blood DC is cytotoxic to human and porcine aortic endothelial cells. Unlike DCs, CD14+ monocytes show no cytotoxicity against the endothelial cells. This cytotoxic potential of IL-15 activated DC against endothelial cells is dose dependent and increases significantly upon treatment of endothelial cells with inflammatory cytokines like TNF-α or IFN-γ. The cytotoxic potential of IL-15 activated DC is associated with apoptosis of endothelial cells, as indicated by the increased Annexin V staining, caspase activation and loss of mitochondrial membrane potential. Further it was observed that DC mediated cytotoxicity against endothelial cell is mediated via granzyme B possibly secreted by the activated DCs.