Conserved features of cancer cells define their sensitivity to HAMLET-induced death; c-Myc and glycolysis.

HAMLET is the first member of a new family of tumoricidal protein-lipid complexes that kill cancer cells broadly, while sparing healthy, differentiated cells. Many and diverse tumor cell types are sensitive to the lethal effect, suggesting that HAMLET identifies and activates conserved death pathways in cancer cells. Here, we investigated the molecular basis for the difference in sensitivity between cancer cells and healthy cells. Using a combination of small-hairpin RNA (shRNA) inhibition, proteomic and metabolomic technology, we identified the c-Myc oncogene as one essential determinant of HAMLET sensitivity. Increased c-Myc expression levels promoted sensitivity to HAMLET and shRNA knockdown of c-Myc suppressed the lethal response, suggesting that oncogenic transformation with c-Myc creates a HAMLET-sensitive phenotype. Furthermore, HAMLET sensitivity was modified by the glycolytic state of tumor cells. Glucose deprivation sensitized tumor cells to HAMLET-induced cell death and in the shRNA screen, hexokinase 1 (HK1), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 and hypoxia-inducible factor 1α modified HAMLET sensitivity. HK1 was shown to bind HAMLET in a protein array containing ∼8000 targets, and HK activity decreased within 15 min of HAMLET treatment, before morphological signs of tumor cell death. In parallel, HAMLET triggered rapid metabolic paralysis in carcinoma cells. Tumor cells were also shown to contain large amounts of oleic acid and its derivatives already after 15 min. The results identify HAMLET as a novel anti-cancer agent that kills tumor cells by exploiting unifying features of cancer cells such as oncogene addiction or the Warburg effect.

Intranasal vaccinations with the trans-sialidase antigen plus CpG Adjuvant induce mucosal immunity protective against conjunctival Trypanosoma cruzi challenges.

Trypanosoma cruzi is an intracellular protozoan parasite capable of infecting through mucosal surfaces. Our laboratory has previously elucidated the anatomical routes of infection after both conjunctival and gastric challenge in mice. We have shown that chronically infected mice develop strong immune responses capable of protecting against subsequent rechallenge with virulent parasites through gastric, conjunctival, and systemic routes of infection. We have also shown that intranasal immunizations with the unique T. cruzi trans-sialidase (TS) antigen protect against gastric and systemic T. cruzi challenge. In the current work we have investigated the ability of purified TS adjuvanted with CpG-containing oligonucleotides to induce immunity against conjunctival T. cruzi challenge. We confirm that intranasal vaccinations with TS plus CpG induce TS-specific T-cell and secretory IgA responses. TS-specific secretory IgA was detectable in the tears of vaccinated mice, the initial body fluid that contacts the parasite during infectious conjunctival exposures. We further show that intranasal vaccinations with TS plus CpG protect against conjunctival T. cruzi challenge, limiting local parasite replication at the site of mucosal invasion and systemic parasite dissemination. We also provide the first direct evidence that mucosal antibodies induced by intranasal TS vaccination can inhibit parasite invasion.

Development of T cell lineages in rat lacrimal glands.

PURPOSE: The purpose of this study was to examine T cell development in rat lacrimal glands, determine whether the thymus is the source of immature T cells in this tissue and compare lacrimal gland T lymphocytes with other T cell subpopulations. METHODS: Mononuclear cells were isolated from lacrimal glands of normal or thymectomized female Fischer 344 rats and stained for flow cytometric analysis. RESULTS: The lacrimal gland T lymphocyte population included large percentages of cells with an activated phenotype and also subpopulations of immature, naive and memory T cells. The numbers of immature (Thy-1(+)) lacrimal gland T cells were unchanged following short-term adult thymectomy. In comparison, spleen had large percentages of naive T cells, only a small subpopulation of activated T cells, and similar percentages of immature (Thy-1(+)) T cells, which were nearly eliminated after thymectomy. Lacrimal gland T cells had small subpopulations of TCRgammadelta(+) and CD8alphaalpha( +) T cells, a large subpopulation of NKT cells and many integrin alphaEbeta7( +) T cells. CONCLUSIONS: Lacrimal gland T cells are composed of a variety of subpopulations whose composition is distinct from splenocytes. The marked reduction of immature splenic T cell percentages eleven days after adult thymectomy indicates that these cells were mostly derived from thymic precursors. In contrast, the unchanged percentages of immature lacrimal gland T cells following thymectomy indicate that they may have an extrathymic source. These studies provide a foundation for further investigation into the cellular basis of lacrimal gland immunobiology.

Lymphocyte lineages at mucosal effector sites: rat salivary glands.

Development of T cell lineages and the role of the thymus as a source of immature T cells in parotid (PG) and submandibular salivary glands (SMG) were studied in Fischer 344 rats using the Thy-1/CD45RC/RT6 expression model. In addition, the phenotypes of salivary gland lymphocytes were compared with other conventional and extrathymic populations. PG mononuclear cells consisted of T cells (38%), B cells (29%), and NK cells (4%). SMG had 19% T cells, 7% B cells, 37% NK cells, and an unusual population of CD3(-)/RT6(+) cells. In comparison with lymph node (LN), both PG and SMG were enriched in immature (Thy-1(+)) and activated (Thy-1(-)/CD45RC(-)/RT6(-)) T cells. Unchanged percentages of Thy-1(+) T cells in PG and SMG following short-term adult thymectomy indicated that immature salivary gland T cells had an extrathymic source. In contrast, thymectomy eliminated LN recent thymic emigrants. SMG had T cells with characteristics of extrathymic populations, expressing TCRgammadelta(+) (28%), the CD8alphaalpha homodimer (11%), and NKR-P1A (66%). Many SMG T cells expressed integrin alpha(E)beta(7). PG T cells resembled those isolated from LN in respect to TCR and CD8 isoform usage, but were enriched in alpha(E)beta(7)(+) T cells and in NKT cells. Thus, salivary gland mononuclear cells are composed of a variety of subpopulations whose distributions differ between SMG and PG and are distinct from LN. These studies provide a basis for further investigation of regionalization in the mucosal immune network and are relevant to the design of vaccine regimens and intervention during pathological immune processes.

Lymphocyte adhesive interactions with cultured parotid salivary gland epithelial cells from rats.

Cellular interactions control lymphocyte localization within salivary gland tissues and contribute to the immune defense of oral surfaces. We examined lymphocyte adherence to cultured parotid cells using an in vitro assay and found good correlation with previously reported binding to parotid gland frozen sections. Thoracic duct lymphocytes (TDL) bound to parotid cells in greater numbers than thymocytes (74 vs 11 cells/mm2). B cells showed preferential adherence compared to T cells (75% vs 28%). TDL binding was inhibited by sodium azide or cytochalasin B (60% and 80%, respectively). EDTA inhibition (63%) was restored by replacing calcium (9%) but not magnesium (65%). Binding was inhibited by fucoidin or phosphomannan (approximately 70%). Fibronectin peptides had no effect. Culture supernatants were inhibitory for TDL adherence (60%), suggesting that molecules involved in lymphocyte localization may be shed and that parotid cell cultures will be useful for ligand isolation and characterization.

Nasal-associated lymphoid tissue is an inductive site for rat tear IgA antibody responses.

The role of nasal-associated lymphoid tissue (NALT) as a mucosal inductive site for tear IgA antibody responses was investigated in the rat model. Fluorescent microspheres were shown to access and be taken up by NALT after intranasal of ocular-topical administration, although fewer microspheres were found in the latter case. Tear IgA anti-DNP antibody responses to dinitrophenylated Streptococcus pneumoniae were 6 micrograms/ml at day 7, 10 micrograms/ml at day 10, and were still detectable on day 21 (5 micrograms/ml) following ocular or gastrointestinal immunization. Intranasal immunization induced tear IgA responses which were 1.7-fold higher at day 7 (10 micrograms/ml), peaked by day 10 (14 micrograms/ml) and were still 1.6-fold higher (8 micrograms/ml) at day 21 than responses of ocular or gastrointestinal groups. These findings suggest that intranasal immunization may be more effective than ocular or gastrointestinal administration in eliciting tear IgA antibody responses and, taken together with the microsphere data, indicate that NALT can serve as an inductive site for ocular mucosal IgA responses.

Phenotypic profiles of lymphocyte populations isolated from rat major salivary glands.

Marker expression was studied in rat lymphocyte populations isolated from parotid, submandibular and sublingual salivary glands. Comparative data were also obtained for lacrimal gland and spleen populations. Increased percentages of Thy-1+ cells were found in salivary gland populations when compared to spleen with the highest percentage noted for parotid gland. Thy-1 percentages in parotid gland were comparable to those obtained with lacrimal gland. Salivary gland sIg, CD5 and CD8 cell percentages were lower than those obtained for lacrimal gland and splenic populations. The percentages of CD4-bearing cells in submandibular and sublingual gland were lower than those found in parotid gland, lacrimal gland and spleen, and the CD4:CD8 ratios in parotid gland most closely approximated those in spleen. Increased percentages of Thy-1+ lymphocytes coexpressing sIg and CD5 were obtained for all salivary gland cell populations when compared to spleen; however, percentages of salivary gland cells bearing these 3 markers were lower than noted for lacrimal gland, which contained the highest percentage. With respect to adhesion molecules, lymph node homing receptor (LNHR) and Peyer's patch homing receptor (PPHR) bearing cells were found in all glandular populations with the percentages of LNHR+ exceeding PPHR+ lymphocytes. Homing receptor bearing populations were highest in spleen and were present in equal proportions. LFA-1 was expressed by all salivary gland cell populations in greater percentages than lacrimal gland, but lower than those in spleen. VLA-4 and CD44 expression was higher in parotid gland and spleen than in submandibular and lacrimal gland. These data show that the phenotypes of resident lymphocytes in salivary gland tissues differ from lacrimal gland and spleen, as well as each other, and indicate that Thy-1+ cells in glandular tissues bear both B and T cell markers. The adhesion molecule expression data suggest that these molecules may be utilized differently in various glandular tissues, potentially contributing to the variation in phenotypic profiles of resident glandular lymphocytes.

The specificity of adhesive interactions between rat lymphocytes and salivary gland epithelia.

Salivary immune responses depend on localization of immunocytes in salivary glands. We tested effects of anti-adhesion molecule antibodies and several ligand analogs on in vitro adherence of rat thoracic duct lymphocytes (TDL) to parotid and submandibular gland sections. While TDL adherence to both tissues was markedly decreased by anti-L-selectin mAbs, binding ability after removal of L-selectin by chymotrypsin or PMA suggested that other adhesion systems were involved. Integrin involvement in parotid interactions was indicated by inhibitory effects of anti-HEBF(PP), LFA-1, ICAM-1, and alpha4 integrin antibodies as well as by the PMA-enhanced adherence. Anti-Thy-1 partially inhibited TDL binding to parotid gland, and anti-CD44 partially inhibited submandibular binding. The majority of salivary gland-bound TDL were sIg+ B cells. FACS analysis showed differences in parotid and submandibular endogenous lymphocyte adhesion molecule expression with greater percentages of L-selectin, HEBF(PP), alpha4 integrin, LFA-1, ICAM-1, CD44, and Thy-1-positive cells present in parotid gland. While precise roles of known or novel adhesion molecules in salivary gland lymphocyte retention are not clear, these data suggest that selectins (parotid, submandibular), integrins (parotid), Thy-1 (parotid), and CD-44 (submandibular), as well as other unidentified molecules, are involved.

Lymphocyte adhesive interactions with lacrimal gland acinar epithelial cells in primary culture.

PURPOSE: In lacrimal glands, cell-cell interactions control the localization of lymphocyte populations that play a role in immune defense at the ocular surface. This study describes lymphocyte adhesive interactions with cultured lacrimal gland acinar epithelial cells. METHODS: Primary cultures of lacrimal gland epithelial cells were used as targets for in vitro lymphocyte binding assays. The relative adherence of lymphocyte populations was determined. Various physiologically active agents and putative ligand analogs were tested for their effect in the binding assay. RESULTS: Thoracic duct lymphocytes (TDL) bound to cultured lacrimal acinar epithelial cells in greater numbers than did thymocytes (54 cells/mm2 versus 8 cells/mm2). B cells showed preferential adherence compared with T cells (75% sIg+, 14% W3/13+). Thoracic duct lymphocyte binding required intact metabolic and membrane-cytoskeletal function and was inhibited by treating the lymphocytes with sodium azide, formaldehyde, or cytochalasin B (23%, 12%, and 10% of control binding, respectively). Further, adherence was dependent on divalent cations. Ethylenediaminetetraacetic acid-mediated inhibition (42% of untreated) was restored by replacing calcium (89%) but not magnesium (41%). Lymphocyte adherence was inhibited in the presence of fucoidin or phosphomannan polysaccharides (36% and 48% of control binding, respectively). Fibronectin peptides, which are involved in certain types of integrin-mediated adherence, had no effect in this system. Lacrimal culture supernatants contained a factor that was inhibitory for TDL adherence (more than 50% inhibition when concentrated 5 or 10 times). CONCLUSIONS: Thoracic duct lymphocyte adherence to cultured lacrimal gland acinar epithelial cells shows good correlation with previously reported adherence to lacrimal gland frozen sections. Further, lacrimal cell culture supernatants contain soluble factors that inhibit TDL adherence to epithelial cells. These findings suggest that the lacrimal molecules involved in lymphocyte localization are shed and that lacrimal epithelial cell cultures will be useful for ligand isolation and characterization.

In vitro adhesive interactions between rat lymphocytes and lacrimal gland acinar epithelium. Phenotype of adherent lymphocytes and involvement of adhesion molecules.

The selective interaction of trafficking lymphocytes with glandular epithelial cells is thought to link the lacrimal gland (LG) to the mucosal immune network. An in vitro binding assay was used to determine the phenotype of adherent cell populations and to study the involvement of lymphocyte adhesion molecules in the adherence process. Enriched B cell populations showed greater binding to LG epithelium than did enriched T cell populations. Direct phenotyping of adherent lymphocytes demonstrated that B lymphocytes (particularly IgA+ and IgG+ cells) were the predominant participants in LG binding. In vitro binding to LG acinar epithelium was inhibited by mAb with specificity for rat lymph node and Peyer's patch homing receptors and, to a lesser degree, by anti-VLA-4 and LFA-1 but not by anti-CD44 or Thy-1. In addition, the presence of rat lymph node and Peyer's patch homing receptors on endogenous LG lymphocytes was demonstrated by flow cytometry. These data show that B cells are the predominant population adhering to LG epithelium and suggest that lymphocyte homing receptors mediate the adherence process. These findings indicate that selective interactions between lymphocytes and the glandular epithelium contribute to the presence of IgA-producing cells in the LG.

Inhibition of lymphocyte adherence to rat lacrimal acinar epithelium by interleukin-4 and transforming growth factor-beta 1.

Mature circulating lymphocyte populations specifically bind to lacrimal gland acinar epithelium in vitro and this adherence is thought to contribute to the accumulation of lymphoid subsets within lacrimal tissue in vivo. The regulatory role of interleukin-4 (IL-4) and transforming growth factor-beta (TGF-beta) in this adherence process was examined using an in vitro binding assay. Pretreatment of thoracic duct lymphocytes (TDLs) with increasing concentrations of IL-4 or TGF-beta for 1 hr resulted in a dose-dependent inhibition of lymphocyte binding to lacrimal gland acinar epithelium. In contrast, the binding of TDLs to high endothelial venules of cervical lymph node was not inhibited by either cytokine. Further, IL-4 and TGF-beta pretreatment did not alter the expression of lymph node or Peyer's patch homing receptors as well as the LFA-1, VLA-4, or CD44 adhesion molecules on TDLs. These results suggest that the interaction of lymphocytes with lacrimal gland acinar epithelium may be regulated by a receptor-mediated mechanism that differs from those governing HEV recognition.

In vitro adherence of rat lymphocytes to salivary gland epithelia.

Glandular mucosal tissues contain lymphocyte populations that contribute to expression of IgA antibodies in external secretions. Interaction of circulating lymphocytes with glandular structures may regulate lymphocyte accumulation. An in vitro assay was used to investigate adhesive interactions between lymphocytes and salivary gland tissues. Thoracic duct lymphocytes (TDL) bound to the serous acinar epithelia of parotid salivary glands and to the mucous tubulo-acinar epithelium of submandibular salivary glands. Lymph node cells and splenocytes adhered to these tissues in lesser numbers and thymocytes bound in negligible numbers. TDL adherence was an active process, being time- and cell dose-dependent and requiring intact membrane as well as cytoskeletal and metabolic function. Calcium was required in each case and binding was mediated by a trypsin-sensitive lymphocyte surface determinant. These findings suggest that the lymphocyte composition of salivary gland tissues is regulated by active lymphocyte interaction with the glandular epithelium.

Selective interactions of lymphocytes with neonatal and adult lacrimal gland tissues.

The lacrimal gland is a functional part of the mucosal immune system and is populated by lymphoid cells that begin to appear early in neonatal development. To define the events controlling the accumulation of these cells, an in vitro adherence assay was used to investigate the interactions of lymphocyte populations with neonatal and adult lacrimal tissue. It was found that (1) lymphocytes adherence to neonatal lacrimal tissue is significantly enhanced over that seen in adults; (2) the increased binding is caused, in part, by adherence to nonacinar structures in neonatal tissue; (3) binding to both neonatal and adult lacrimal gland tissue is an active process that is observed only with viable lymphocytes; (4) lymphocyte adherence to neonatal and adult lacrimal gland tissues is differentially affected by metabolic and cytoskeletal inhibitors; and (5) attachment appears to require the presence of Ca++, a lymphocyte surface protein, and may involve target tissue carbohydrate recognition. These findings suggest that the initial accumulation of lymphocytes in the neonatal lacrimal gland results from a generalized enhanced binding capacity of the developing tissue and that the preferential binding of certain populations (thoracic duct and mesenteric lymph node lymphocytes) to acinar cells maintains the pool in the adult lacrimal gland.