DNAM-1 controls NK cell activation via an ITT-like motif

DNAM-1 (CD226) is an activating receptor expressed on natural killer (NK) cells, CD8⁺ T cells, and other immune cells. Upon recognition of its ligands, CD155 and CD112, DNAM-1 promotes NK cell–mediated elimination of transformed and virus-infected cells. It also has a key role in expansion and maintenance of virus-specific memory NK cells. Herein, the mechanism by which DNAM-1 controls NK cell–mediated cytotoxicity and cytokine production was elucidated. Cytotoxicity and cytokine production triggered by DNAM-1 were mediated via a conserved tyrosine- and asparagine-based motif in the cytoplasmic domain of DNAM-1. Upon phosphorylation by Src kinases, this motif enabled binding of DNAM-1 to adaptor Grb2, leading to activation of enzymes Vav-1, phosphatidylinositol 3′ kinase, and phospholipase C-γ1. It also promoted activation of kinases Erk and Akt, and calcium fluxes. Although, as reported, DNAM-1 promoted adhesion, this function was signal-independent and insufficient to promote cytotoxicity. DNAM-1 signaling was also required to enhance cytotoxicity, by increasing actin polymerization and granule polarization. We propose that DNAM-1 promotes NK cell activation via an immunoreceptor tyrosine tail (ITT)–like motif coupling DNAM-1 to Grb2 and other downstream effectors.DNAX accessory molecule-1 (DNAM-1), also known as CD226, is a receptor expressed on natural killer (NK) cells, CD8⁺ T cells, some CD4⁺ T cells, and some myeloid cells (Shibuya et al., 1996; Long et al., 2013; de Andrade et al., 2014; Martinet and Smyth, 2015). Although it is part of the Ig superfamily, DNAM-1 is rather unique. This uniqueness is especially evident in the cytoplasmic domain, which shares little or no homology with other Ig superfamily members. DNAM-1 recognizes CD155 (poliovirus receptor) and CD112 (nectin-2) as ligands, which are expressed on a broad range of cells, including transformed cells and virus-infected cells (Bottino et al., 2003). CD155, CD112, or both are also ligands for the inhibitory receptors TIGIT and CD96 (tactile), which are also expressed on immune cells. As loss of DNAM-1 enhances the availability of CD155 and CD112 for TIGIT and CD96, this feature complicates interpretation of phenotypes found in mice lacking DNAM-1.DNAM-1 was initially identified as a molecule promoting cytotoxicity and cytokine secretion by NK cells and CD8⁺ T cells (Shibuya et al., 1996). Subsequent work revealed that DNAM-1 was important for NK cell–mediated killing of tumor cells such as melanoma cells, rhabdomyosarcoma cells, and Ewing’s sarcoma cells (Verhoeven et al., 2008; Lakshmikanth et al., 2009; Cho et al., 2010). Accordingly, DNAM-1–deficient mice were more susceptible to carcinogen-induced fibrosarcoma and papilloma in vivo (Gilfillan et al., 2008; Iguchi-Manaka et al., 2008). DNAM-1 was also implicated in NK cell–mediated elimination of HIV-infected CD4⁺ T cells and human cytomegalovirus (HCMV)-infected DCs (Magri et al., 2011; Matusali et al., 2012). Moreover, in mice, it played a critical role in expansion and survival of virus-specific memory NK cells in mouse cytomegalovirus (MCMV)–infected mice (Nabekura et al., 2014). A key role of DNAM-1 was also documented in CD8⁺ T cells. DNAM-1 was critical for the ability of CD8⁺ T cells to eliminate tumor cells and virus-infected cells during blocking antibody therapy targeting the inhibitory receptor TIGIT (Johnston et al., 2014). Likewise, DNAM-1 was necessary for the ability of CD8⁺ T cells to mediate acute graft-versus-host disease in mice (Nabekura et al., 2010).Early studies suggested that human DNAM-1 promotes NK cell activation, at least in part, by acting as an adhesion receptor, which stabilizes physical contacts between NK cells and target cells (Shibuya et al., 1996, 1999). This function was reportedly dependent on the ability of DNAM-1 to bind in cis to integrin LFA-1. DNAM-1 was also shown to undergo phosphorylation at a conserved tyrosine (Y) in its cytoplasmic domain (Y319 in mouse and Y322 in human; Shibuya et al., 1999). This phosphorylation was reported to be mediated by Src family kinase Fyn. Although the precise role of Y319 was not elucidated (for simplification, mouse numbering will be used in this report), the ability of DNAM-1 to stimulate accumulation of virus-induced memory-like NK cells in mice was dependent on Y319 (Nabekura et al., 2014). Human DNAM-1 was also described to undergo phosphorylation at serine 326 (S326) in its cytoplasmic domain, as a result of the action of protein kinase C (Shibuya et al., 1998, 1999). This phosphorylation was reported to promote the DNAM-1–LFA-1 association and, in mice, to be critical for accumulation of memory-like NK cells in virus-infected mice (Shibuya et al., 1999; Nabekura et al., 2014).Various reports have examined the possibility that DNAM-1 transduces intrinsic biochemical signals. In some studies, engagement of human DNAM-1 by anti–DNAM-1 antibodies failed to trigger activation of kinases Erk and Akt or calcium fluxes (Bryceson et al., 2006; Chen et al., 2007). In other studies, human DNAM-1 synergized with 2B4 to enhance tyrosine phosphorylation of adaptor SLP-76 and exchange factor Vav-1 (Kim and Long, 2012). This effect correlated with increased NK cell–mediated cytotoxicity and cytokine production. However, the mechanism by which DNAM-1 influenced these signals was not determined.Whether DNAM-1 functions as an adhesion receptor, a signal transduction molecule or both during NK cell activation needs to be resolved. The structural basis for the ability of DNAM-1 to mediate possible signals, as well as the effectors of these signals, also requires clarification. Lastly, the cellular mechanism by which DNAM-1 promotes NK cell activation, i.e., enhanced cellular adhesion or other mechanisms, requires clarification.Herein, we addressed these issues using a combination of genetic, biochemical, molecular, and imaging approaches. These studies showed that engagement of DNAM-1 triggered active signals, which were required for the ability to promote NK cell activation. These signals were initiated by Y319, but not S326, of DNAM-1. They also necessitated an asparagine at position 321 (N321), which cooperated with phosphorylated Y319 to bind adaptor Grb2. Binding of DNAM-1 to Grb2 enabled activation of exchange factor Vav-1, phosphatidylinositol (PI) 3′ kinase, and phospholipase C (PLC)-γ1. DNAM-1 signaling did not enhance cytotoxicity by promoting adhesion. Rather, it acted by enhancing actin polymerization and polarization of lytic granules toward target cells.     

Insulin-like growth factor-I can mediate autocrine proliferation of human small cell lung cancer cell lines in vitro.

The effect of insulin-like growth factor I (IGF-I) on growth of small cell lung cancer (SCLC) cell lines was studied. Western blot analysis of whole cell lysates of cell lines NCI-H345 and NCI-N417 demonstrated the presence of a 16-kD band consistent with an IGF-I precursor molecule. Scatchard plot analysis of cell line NCI-H345 using 125I-labeled IGF-I demonstrated two high affinity specific binding sites (Kd 1.3 and 4.0 nM with maximal rate (Bmax) 200 and 500 fmol/mg protein, respectively). The exogenous addition of IGF-I, IGF-II, or insulin resulted in marked proliferation of human SCLC cells as evaluated using an in vitro growth assay. These peptides stimulated the growth of SCLC cell lines NCI-H82, NCI-H209, NCI-H345, and NCI-N417. The concentration of IGF-I producing maximal SCLC cell growth was 10-100-fold less than that of insulin or IGF-II, whereas the maximal growth stimulated by the optimal concentration of these peptides were similar. An MAb that specifically binds to the IGF-I receptor (but not to the insulin receptor) mediates a dose-dependent inhibition of cell growth in basal media as well as IGF-I, IGF-II, or insulin-supplemented media. The IGF-I receptor thus appears to be the common pathway for the mitogenic activity by IGF-I, IGF-II, and insulin for human SCLC cell lines. The demonstration of an IGF-I precursor molecule, specific IGF-I receptor binding, IGF-I-mediated growth stimulation, and inhibition of basal cell growth by an MAb to the IGF-I receptor suggests that an IGF-I-like molecule can function in vitro as an autocrine growth factor for human SCLC cell lines.     

IL-15 trans-presentation promotes human NK cell development and differentiation in vivo

The in vivo requirements for human natural killer (NK) cell development and differentiation into cytotoxic effectors expressing inhibitory receptors for self–major histocompatability complex class I (MHC-I; killer Ig-like receptors [KIRs]) remain undefined. Here, we dissect the role of interleukin (IL)-15 in human NK cell development using Rag2^−/−γc^−/− mice transplanted with human hematopoietic stem cells. Human NK cell reconstitution was intrinsically low in this model because of the poor reactivity to mouse IL-15. Although exogenous human IL-15 (hIL-15) alone made little improvement, IL-15 coupled to IL-15 receptor α (IL-15Rα) significantly augmented human NK cells. IL-15–IL-15Rα complexes induced extensive NK cell proliferation and differentiation, resulting in accumulation of CD16⁺KIR⁺ NK cells, which was not uniquely dependent on enhanced survival or preferential responsiveness of this subset to IL-15. Human NK cell differentiation in vivo required hIL-15 and progressed in a linear fashion from CD56^hiCD16⁻KIR⁻ to CD56^loCD16⁺KIR⁻, and finally to CD56^loCD16⁺KIR⁺. These data provide the first evidence that IL-15 trans-presentation regulates human NK cell homeostasis. Use of hIL-15 receptor agonists generates a robust humanized immune system model to study human NK cells in vivo. IL-15 receptor agonists may provide therapeutic tools to improve NK cell reconstitution after bone marrow transplants, enhance graft versus leukemia effects, and increase the pool of IL-15–responsive cells during immunotherapy strategies.NK cells participate in host protection by eliminating cells with altered expression of self–MHC class I (MHC-I), which can result from viral infection or transformation (1). Although we are beginning to appreciate a role for viral and stress-induced ligands in NK cell activation, the best described regulatory mechanism of NK cell activity is the expression of inhibitory receptors for self–MHC-I ligands by mature NK cells with high cytotoxic potential. In man, killer Ig-like receptors (KIRs) recognizing the classical MHC-I molecules HLA-A, -B, or -C are expressed on the predominate peripheral NK cell (CD56^loCD16⁺) subset, which possesses abundant intracellular perforin and granzymes and displays spontaneous cytotoxicity (1). In contrast, CD56^hiCD16⁻ NK cells rarely express KIRs, and because they are more prevalent in blood early after bone marrow transplant (2, 3), give rise to CD56^loCD16⁺ NK cells when transferred into NOD/SCID mice (4), and have longer telomeres than CD56^loCD16⁺ NK cells (5), it is likely that CD56^hiCD16⁻ NK cells are (or contain within this population) precursors of CD56^loCD16⁺ NK cells; however, the tools to definitively prove this hypothesis are lacking.Although inhibitory KIRs control reactivity of mature NK cells, their expression also influences the functional maturation of developing NK cells, as NK cells expressing at least one KIR-recognizing self–MHC-I have a lower threshold of activation and appear more functional than NK cells expressing no KIRs or those only expressing KIRs recognizing non-self–MHC-I ligands (6). In addition, patients lacking the transporter associated with antigen processing have dramatically reduced MHC-I surface expression and, consequently, hyporesponsive NK cells to MHC-I–deficient cells (7). KIR⁺ NK cells are present in these patients, indicating that normal MHC-I expression itself is not required for KIR expression (7). This phenomenon termed “licensing” or “disarming” has been better characterized in mice, and suggests a role for KIR–self–MHC-I interactions during human NK cell development. Given the importance of KIR expression in regulating NK cell function, knowledge of elements influencing KIR acquisition would improve our understanding and clinical approaches to diseases where KIR and HLA haplotypes influence susceptibility, progression, or outcome of autoimmune/inflammatory disease, cancer, hematopoietic grafts, and infections (8).Because KIRs are expressed on mature CD56^loCD16⁺ NK cells, factors that influence NK cell homeostasis could potentially influence KIR acquisition in vivo. An elegant series of mouse studies using gene targeting and bone marrow chimeras have revealed that NK cell development requires IL-15Rα–expressing cells to chaperone IL-15 to the surface, where it is bioactive and significantly more potent in inducing activation and proliferation of IL-15Rβγ–expressing cells via trans-presentation (9, 10). When not bound to IL-15Rα, IL-15 appears to have a minimal effect on NK cell homeostasis in vivo (11, 12). In humans, mutations in IL-15Rα have not been reported; however, NK cells are dramatically reduced in patients carrying mutations in the common γ chain (γc) cytokine receptor (used in IL-15, -7, -4, -9, -2, and -21 signal transduction), Jak3, or the shared IL-2/-15Rβ, whereas they are present in IL-7Rα–deficient patients, suggesting that IL-15 may regulate human NK cell development (13, 14).In vivo studies of NK cells have been largely restricted to mice, and although this line of experimentation is valuable, some of this knowledge is not transferable to human NK cell biology. A clear example of this is NK cell development, where the kinetics, frequency, and phenotype are clearly different between species (1). An intermediate between mouse and human in vivo studies exists in the form of human immune system (HIS) mice. A recently developed HIS mouse model is the engraftment of newborn BALB/c Rag2^−/−γc^−/− mice with human hematopoietic stem cells (HSCs) from fetal liver or cord blood (15, 16). BALB/c Rag2^−/−γc^−/− HIS mice represent a practical HIS model with high human chimerism, most lymphocyte lineages generated, and adaptive immune responses occasionally evoked (15, 16).In this study, using HIS BALB/c Rag2^−/−γc^−/− mice engrafted with fetal liver HSCs, we study the in vivo role of IL-15 trans-presentation in human NK cell homeostasis.     

Ultrasonic irradiation of mouse tissues in vivo and human amniotic cells in vitro

Recently published results have been substantiated by enlarging the group sizes of mice subjected to whole-body exposure for 200s and of human amniotic cells in vitro for 2000s at 34°C. Spatial average intensity was 1 W/cm². Cyclic AMP, cyclic GMP and histamine in mouse skin, lung and peritoneal cells, and cAMP and cGMP in human amniotic cells were assayed as sensitive indicators of membrane damage. No significant effect of ultrasound was observed. Statistical analysis indicates an 80% probability that we would have detected a difference in the means for exposed and sham exposed groups if it had been greater than one standard deviation in 9 out of 18 comparisons for mouse tissues and 7 out of 8 comparisons for human amniotic cells.     

Spontaneous pyrogen production by mouse histiocytic and myelomonocytic tumor cell lines in vitro.

Tumor-associated fever occurs commonly in acute leukemias and lymphomas. We investigated the capacity for in vitro production of pyrogen by three mouse histiocytic lymphoma cell lines (J-774, PU5-1.8, p 388 D1), one myelomonoyctic line (WEHI-3), and tow lymphoma-derived lines, RAW-8 and R-8. Pyrogen was released spontaneously into the culture medium during growth by all cell lines with macrophage or myeloid characteristics including lysozyme production; R-8 cells, of presumed B-lymphocyte origin, did not produce pyrogen. When injected into mice, the pyrogens gave fever curves typical of endogenous pyrogen, were inactived by heating to 56 degrees C and by pronase digestion, and appeared to be secreted continuously by viable cells. Two pyrogenic molecular species produced by H-774 cells were identified by Sephadex filtration, one of mol wt approximately equal to 30,000, and the other greater than or equal to 60,000. By contrast, three carcinoma cell lines of human origin and SV-40 3T3 mouse fibroblasts did not produce pyrogen in vitro. These results suggest that some malignant cells derived from phagocytic cells of bone marrow origin retain their capacity for pyrogen production, and may spontaneously secrete pyrogen during growth.     

体外培养人黄韧带细胞系的建立及细胞学特性分析

目的为研究黄韧带骨化致椎管狭窄的机制打下基础.方法通过体外组织块培养的方法,建立起正常、退化和骨化黄韧带的细胞系共18株,对细胞的生长特点进行了初步分析.结果培养的黄韧带细胞可以在体外增殖和传代,细胞呈现多种形态,以梭形为主,特别是退变和骨化患者的韧带细胞可形成钙化结节.结论首次成功的培养出黄韧带细胞,为深入研究黄韧带骨化的相关机制提供一种有力的手段.     

The immunobiological properties expressed in vitro and in vivo of Salmonella enteritidis-induced murine T cell lines

Splenic T lymphocytes from two strains of mice, BALB/c and B10.BR, infected with an attenuated strain of Salmonella enteritidis were cloned by the double-layer soft agar technique in the presence of interleukin 2 (IL 2), formalin-killed S. enteritidis (FKS) and syngeneic feeder cells. One Salmonella-reactive T cell line was established from each strain of mice. Both T cell lines bore Thy-1+, Lyt-1+ and L3T4+ surface markers as demonstrated by cytofluorography. Biological properties of the T cell lines were studied with respect to their ability to proliferate and produce lymphokines such as IL 2 and gamma-interferon (IFN-gamma) in response to Salmonella antigens, and to transfer adoptively protection against infection and delayed-type hypersensitivity (DTH). As the result of the present study, the T cell lines were proliferated specifically against several Salmonella and other bacteria, which belong to species of Enterobacteriaceae. Their proliferation required the presence of the specific antigen(s) and the compatibility in the I-A region of the H-2 complex between the T cell lines and feeder cells. The T cell lines could be proliferated with resultant production of IL 2 and IFN-gamma by in vitro culture in the presence of syngeneic feeder cells and Salmonella antigens. The protective activity assessed by the number of recoverable bacteria in spleens and livers after challenge with virulent S. enteritidis and DTH reactions to Salmonella antigen were exhibited by the T cell lines when transferred adoptively to naive syngeneic mice. These results suggested that different biological functions of cell-mediated immunity to Salmonella could be mediated by a single phenotype of T cell population.     

Steroid hormone enhancement of gene delivery to a human airway epithelial cell line in vitro and mouse airways in vivo.

Current liposome-based delivery protocols for gene therapy are relatively inefficient. In a pharmacological approach to enhance liposome-mediated gene delivery we have evaluated beta-estradiol and methyl-prednisolone as enhancing agents. We have shown that beta-estradiol in combination with lipoplex can significantly increase luciferase gene expression in sub-confluent, confluent and polarized human bronchial epithelial (16HBE) cells 23-fold, 100-fold and 900-fold, respectively, when compared with lipoplex alone. Similarly, incorporation of methyl-prednisolone into lipoplexes increases luciferase gene expression in confluent and polarized 16HBE cells 70.8-fold and 48-fold, respectively. Greater levels of gene expression were obtained when beta-estradiol (9.5-fold enhancement) or methyl-prednisolone (14-fold enhancement) were mixed with the liposome before addition of the plasmid compared with addition of the steroid after lipoplex formation. Beta-estradiol-containing lipoplexes were also evaluated in vivo where in the murine lung and nasal epithelium an eight-fold and 7.5-fold enhancement in gene expression were found compared with lipoplex alone. Incorporating beta-estradiol into lipoplexes increased both the total number of cells transfected and the amount of intracellular plasmid within the cell, including the nuclear compartment, compared with lipoplex alone. These results demonstrate the ability of steroids to enhance gene delivery in vitro and in vivo and thus may have the potential to improve gene therapy strategies.     

四种人胃癌细胞体外增殖和侵袭能力比较

目的比较四种不同组织来源、不同分化程度的胃癌细胞MGC-803、HGC-27、BGC-823、SGC-7901的体外增殖和侵袭能力。方法分别培养四种细胞,用直接计数法绘制细胞生长曲线,计算其群体倍增时间;以细胞克隆形成率比较其增殖能力;体外划痕法和Transwells法比较四种细胞迁移、侵袭能力。结果与MGC-803和HGC-27相比,BGC-823和SGC-7901的生长速度依次减慢,群体倍增时间依次增加(P<0.05);平板克隆形成率从MGC-803、HGC-27、BGC-823到SGC-7901分别是42.7±2.2、38.6±1.6、28.9±1.7、21.3±1.9,差异有统计学意义(P<0.05)。体外划痕法和Transwells小室侵袭实验均表明MGC-803和HGC-27细胞的迁移侵袭能力较BGC-823和SGC-7901高,差异有统计学意义(P<0.05)。结论四株胃癌细胞株,从MGC-803、HGC-27、BGC-823到SGC-7901其增殖和侵袭能力逐渐下降。     

Dual-pulse lithotripter accelerates stone fragmentation and reduces cell lysis in vitro

Lithotripsy is a common effective treatment for kidney stones. However, focal volumes are often larger than stones, and surrounding tissue is often injured. Our goal was to test in vitro a new lithotripter consisting of two opposing, confocal and simultaneously triggered electrohydraulic sources. The pulses superimpose at the common focus, resulting in pressure doubling and enhanced cavitation growth in a localized, approximately 1-cm wide volume. Model gypsum stones and human erythrocytes were exposed to dual pulses or single pulses. At the focus, model stones treated with 100 dual pulses at a charging voltage of 15 kV broke into 8 times the number of fragments as stones treated with 200 single pulses at 18 kV. At axial positions 2 and 4 cm away from the focus, lysis of erythrocytes was reduced or equivalent for dual pulses vs. single pulses. Hence, in half the time, dual pulses increased comminution at the focus without increasing injury in surrounding regions.     

Interleukin 2 inhibits in vitro growth of human T cell lines carrying retrovirus

Four human T cell lines, TL-Mor, TL-Su, TL-TerI, and TL-OmI, carrying human T cell leukemia virus (HTLV), were established previously. TL-Mor, TL-Su, and TL-TerI were derived from interleukin 2 (IL-2)-dependent parental cell lines cloned from peripheral blood leukocytes (PBL) of three healthy HTLV carriers, while TL-OmI was directly established from PBL of a patient with adult T cell leukemia. These four TL cell lines grow autonomously without IL-2. When they were cultured in the presence of IL-2, their growth was inhibited after a few days. This growth inhibition depended on the dose of IL-2, and the effective dose significantly promoted growth of their parental IL-2-dependent cell lines. The growth inhibition is demonstrated to be due to specific binding of IL-2 to receptors on the TL cells.     

Induction of in vitro and in vivo NK cell cytotoxicity using high-avidity immunoligands targeting prostate-specific membrane antigen in prostate carcinoma

Cancer that might develop as host natural killer (NK) cells fail to detect ligands for their activating NK receptors. Immunoligands represent promising immunotherapeutic tools to overcome this deficit. These are fusion proteins containing a single-chain antibody fragment (scFv) to target an available tumor antigen and ULBP2 to activate host NK cells by targeting the activatory receptor NKG2D. Prostate-specific membrane antigen (PSMA) is an integral non-shed type 2 membrane protein that is highly and specifically expressed on prostate epithelial cells and strongly upregulated in prostate cancer. Here, we compare the impact of various anti-PSMA immunoligand formats on the therapeutic efficacy against prostate carcinoma cells by activating NK cells via NKG2D. Shortening of the linker separating the heavy and light chain antibody domain leads to the formation of dimers, trimers, and higher molecular mass oligomers. NK cells are most efficiently activated by multimeric immunoligands, thus showing an altered cytokine release pattern. The high avidity format is also superior in in vitro NK-mediated tumor cell targeting as shown in cytotoxicity assays. Finally, the efficacy of a multimeric immunoligand is shown in a prostate carcinoma mouse xenograft model showing a strong activity against advanced established tumors.     

Effector T cell analysis of melanoma tumor-infiltrating lymphocyte cultures using HLA-ABC semimatched melanoma cell lines

The generation of T cells with specific reactivity against tumor-associated antigens is prerequisite for adoptive transfer therapy. Melanoma-specific lymphocyte cultures can be established from tumor-infiltrating lymphocytes (TILs) by in vitro culture with high levels of interleukin-2. In this report, we present TIL data originating from 728 attempted cultures from 33 consecutive melanoma biopsy specimens originating from 30 patients. Cultures were analyzed for the presence of interferon gamma (IFNgamma)-producing cells upon stimulation with a panel of HLA-ABC semimatched melanoma cell lines. We sought to find whether such cell lines could be used to analyze TIL reactivity. Cell lines were used as stimulators to circumvent the need for autologous primary tumor cells. Melanoma-reactive cultures were identified by flow cytometry in 25 of the 30 patients. Four hundred forty-four of 728 (60.9%) cultures contained TILs at the end of experiment. Ninety-one of 318 cultures (28.6%) contained IFNgamma-producing cells after stimulation. In HLA-A*0201 patients IFNgamma analysis was complemented with melanoma-specific tetramer staining. All but one HLA-A*0201 patient had MART-1/Melan-A27-35-directed TILs, with frequencies ranging from 0.1% to 90% of CD8 cells. In addition, tetramer analysis also identified TILs directed against gp100, Tyrosinase, and Her2Neu. Tumor material was collected via needle biopsy in 16 cases and surgery in 18 cases. Overall, surgical material generated more cultures positive for T cells. The described methods are efficient in characterizing clinically relevant melanoma-reactive TILs.     

Establishment of mouse embryonic fibroblast cell lines that promote ex vivo expansion of human cord blood CD34+ hematopoietic progenitors

The development of culture systems that facilitate ex vivo maintenance and expansion of transplantable hematopoietic progenitor cells (HPC) is vital to stem cell transplantation. The use of a monolayer of stromal cells on which to grow HPC in direct contact allows high efficiency ex vivo expansion of HPC. Here, we report an establishment of three murine embryonic fibroblast stromal cell lines from adherent cells of day-12 mouse embryos. Among them, HYMEQ-5 was most efficient in supporting long-term maintenance of human umbilical cord blood (CB) CD34(+) cells. Human CB CD34(+) cells cultured on HYMEQ-5 in the presence of stem cell factor (SCF), thrombopoietin, and flk-ligand (FL) showed high expansion of CD34(+)CD38(-) cells and highly proliferative potential-colony forming cells (HPP-CFC). Direct cell-to-cell contact between CD34(+) cells and HYMEQ-5 was important for this expansion. RT-PCR analysis showed that HYMEQ-5 produced FL, SCF, interleukin-6, and macrophage colony-stimulating factor (M-CSF). Expanded CB CD34(+) cells efficiently reconstituted hematopoiesis in nonobese diabetic/severe combined immunodeficient disease (NOD/SCID) mice. These findings suggest that HYMEQ-5 provides a milieu that supports long-term human hematopoiesis as well as ex vivo expansion of human CB CD34(+) HPC. This cell line may facilitate elucidation of the mechanism of cellular interactions between HPC and stromal cells.     

Cell retargeting by bispecific monoclonal antibodies. Evidence of bypass of intratumor susceptibility to cell lysis in human melanoma.

Intratumor heterogeneity for susceptibility to cytotoxic T lymphocytes (CTL)-mediated lysis represents a major obstacle to cancer adoptive immunotherapy. To overcome the heterogeneity observed in terms of susceptibility of target cells to cell-mediated lysis, in this study we used two purified bispecific monoclonal antibodies (bsmAbs) that recognize molecules expressed by cytotoxic effector cells (CD3 and IgG Fc receptorial molecules), as well as one high molecular weight melanoma-associated antigen (HMW-MAA). The ability of these reagents to enhance or induce a relevant in vitro cytotoxic activity by a CTL clone (CTL 49) isolated from PBL of a melanoma patient was tested on a large panel of autologous and allogeneic melanoma cell lines and clones. Functional studies revealed that the CTL 49 clone lysed all the HMW-MAA+ tumor lines in the presence of bsmAbs and that these reagents affected the target lysis in a cooperative fashion. The effectiveness of bsmAbs in overcoming the heterogeneous susceptibility of human melanoma cells to cell-mediated lysis may find practical implications in cancer adoptive immunotherapy.     

The microbiome restrains melanoma bone growth by promoting intestinal NK and Th1 cell homing to bone

Bone metastases are frequent complications of malignant melanoma leading to reduced quality of life and significant morbidity. Regulation of immune cells by the gut microbiome influences cancer progression, but the role of the microbiome in tumor growth in bone is unknown. Using intracardiac or intratibial injections of B16-F10 melanoma cells into mice, we showed that gut microbiome depletion by broad-spectrum antibiotics accelerated intraosseous tumor growth and osteolysis. Microbiome depletion blunted melanoma-induced expansion of intestinal NK cells and Th1 cells and their migration from the gut to tumor-bearing bones. Demonstrating the functional relevance of immune cell trafficking from the gut to the bone marrow (BM) in bone metastasis, blockade of S1P-mediated intestinal egress of NK and Th1 cells, or inhibition of their CXCR3/CXCL9-mediated influx into the BM, prevented the expansion of BM NK and Th1 cells and accelerated tumor growth and osteolysis. Using a mouse model, this study revealed mechanisms of microbiota-mediated gut-bone crosstalk that are relevant to the immunological restraint of melanoma metastasis and tumor growth in bone. Microbiome modifications induced by antibiotics might have negative clinical consequences in patients with melanoma.     

In vivo and in vitro effect of progesterone on the growth of some mouse and human tumours

The effect of progesterone on the growth of tumours of different morphological and cytokinetic characteristics and origin has been investigated, such as mammary aplastic carcinoma, Ehrlich ascitic and solid tumour, fibrosarcoma, melanoma B-16, myeloid leukaemia and three cell lines, HeLa, HEp-2 and L929. The administration of progesterone has been proved to stimulate the growth of aplastic carcinoma of the breast in vivo. Directly implemented into cell culture of the same tumour it increased the incorporation of 3H-thymidine into DNA. In HeLa and HEp-2 cell cultures progesterone stimulated the population growth of these cells. However, progesterone has shown no effect at all on the growth of fibrosarcoma, melanoma, Ehrlich tumour, myeloid leukaemia and L929 cells, fibroblasts of C3H mice.