Anticlonotypic monoclonal antibodies induce proliferation of clonotype-positive T cells in peripheral blood human T lymphocytes. Evidence for a phenotypic (T4/T8) heterogeneity of the clonotype-positive proliferating cells.

Three previously selected monoclonal antibodies (mAb) directed against the clonotypic structure of a variant (termed JA3) of the interleukin 2 (IL-2)-producing Jurkat leukemia cell line (anti-JTi1-3 mAb) were found to induce an adherent cell-dependent proliferation of peripheral blood T cells in 20 different donors. Unlike the early cell proliferation induced by anti-T3 mAb, anti-JTi mAb-induced proliferation was detectable at day 5-6 of culture and reached peak levels at day 7-9. Less than 1% JTi+ cells were consistently detected in the starting peripheral blood lymphocytes or in control cultures in which cells were stimulated with anti-T3, phytohemagglutinin, or allogeneic cells. However, JTi+ cells were found in increasing proportions after culture with anti-JTi mAb and they were mostly represented by large blast cells expressing either the T4 or the T8 antigen, together with typical activation antigens including HLA-DR, IL-2 receptor, and 4F2. Immunoprecipitation experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that anti-JTi-reactive molecules present on antibody-stimulated lymphocytes or on JA3 cells were similar, disulphide-linked heterodimeric structures.

Quantitative assessment of the pool size and subset distribution of cytolytic T lymphocytes within human resting or alloactivated peripheral blood T cell populations.

In order to directly assess the distribution of cytolytic T lymphocytes (CTL) and their precursors (CTL-P) in the two major subsets of human T cells, we have used limiting dilution microculture systems to determine their frequencies. The two subsets were defined according to their reactivity (or lack thereof) with B9.4 monoclonal antibody (the specificity of which is similar, if not identical, to that of Leu 2b monoclonal antibody). Both B9+ and B9- cells obtained by sorting peripheral blood resting T cells using the fluorescence-activated cell sorter (FACS) were assayed for total CTL-P frequencies in a microculture system that allows clonal growth of every T cell. As assessed by a lectin-dependent assay, approximately 30% of peripheral blood T cells were CTP-P. In the B9+ subset (which represents 20-30% of all T cells), the CTL-P frequency was close to 100%, whereas the B9- subset had a 25-fold lower CTL-P frequency. It is thus evident that 90% and 10% of the total CTL-P in peripheral blood are confined to the B9+ or B9- T cell subsets, respectively. Analysis of the subset distribution of CTL-P directed against a given set of alloantigens confirmed these findings. CTL-P frequencies were also determined in B9+ and B9- subsets derived from T cells that had been activated in allogenic mixed leucocyte cultures (MLC). Approximately 10% of MLC T cells were CTL-P. This frequency was increased 3.5-fold in the B9+ subset, whereas the B9- subset contained only a small, although detectable number of CTL-P. Moreover, the great majority of the (operationally defined) CTL-P in MLC T cell population were found to be directed against the stimulating alloantigens, thus indicating a dramatic increase in specific CTL-P frequencies following in vitro stimulation in bulk cultures.

Surface markers of cloned human T cells with various cytolytic activities.

Human T cells stimulated in secondary allogeneic mixed lymphocyte culture (MLC) were cloned under limiting conditions in microculture systems using T cell growth factor and irradiated allogeneic cells. Clones with lytic activity against either phytohemagglutinin-induced blast cells bearing the stimulating alloantigen(s) (cytotoxic T lymphocyte [CTL] activity), L1210 mouse lymphoma cells coated with rabbit antibody (antibody-dependent cell-mediated cytotoxicity [ADCC]), or K562 human target cells were selected, expanded, and then analyzed for different surface markers, including rosette formation with sheep erythrocytes (E rosettes), receptors for the fc portion of IgG or IgM (Fc gamma R and Fc mu R), and a group of antigens recognized by monoclonal antibodies including Ia, 4F2, OKT8,a nd OKT4. All the cytotoxic cells were E rosette+, Ia+ and 4f2+. Expression of Fc gamma R was restricted to the clones active in ADCC. CTL clones were either OKT8+ or OKT8-. Furthermore, three of the OKT8- CTL clones were OKT4+. In addition, some cytolytic clones devoid of specific CTL activity were OKT8+. It thus appears that the claim that human CTL are OKT8+, OKT4-, and Ia- is not supported by the analysis of their phenotype at the clonal level.

Direct demonstration of the clonogenic potential of every human peripheral blood T cell. Clonal analysis of HLA-DR expression and cytolytic activity.

In an attempt to determine the clonogenic properties of human peripheral blood T cells, we have developed a limiting dilution microculture system using phytohemagglutinin (PHA) as T cell activator and supernatant from PHA-stimulated spleen cultures as a source of T cell growth factors. The frequencies of cells capable of extensive proliferation under these culture conditions were 0.52-0.73, 0.98-1.11, and less than 0.02 in peripheral blood mononuclear, E-rosette-positive, and E-rosette-negative cell populations, respectively. The clonogenic potential of virtually all T cells was confirmed in experiments using single cells isolated by micromanipulation. Clone size ranged between 5 and 30 X 10(4) cells on day 14 of culture. The same microculture system was used to determine the precursor frequency of all cytolytic T lymphocytes (CTL-P). As assessed by a lectin-dependent 51Cr release assay, the CTL-P frequency in purified T cell populations ranged between 0.30 and 0.34. In comparison, the precursor frequency of T cells capable of lysing K562 target cells was ranging between 0.14 and 0.16. Parallel analysis of individual clonal cultures for both lytic activities showed that 50% of the clones exhibiting lectin-dependent lysis were also active against K562 target cells. All of the proliferating clones expressed HLA-DR antigens, although to a varying degree as assessed by flow cytofluorometry. Given the high cloning efficiency of this culture system, it appears now possible to determine the precursor frequencies of the various classes of functional cells in T cell populations.

Clonal heterogeneity in the requirement for T3, T4, and T8 molecules in human cytolytic T lymphocyte function.

In an attempt to define the requirement of T8, T4, and T3 surface molecules in functional interactions occurring between human cytolytic T lymphocytes (CTL) and specific target cells, we have analyzed a large number of CTL clones derived from primary mixed lymphocyte culture (MLC) T cell populations for their susceptibility to inhibition by monoclonal antibodies (mAb) directed against these surface antigens. In most experiments, MLC T cells were stained with B9.4 (anti-T8) or OKT4 (anti-T4) mAb, separated into positive and negative cells using a fluorescence-activated cell sorter (FACS) and cloned under limiting conditions. While the lytic activity of the majority of T8+ CTL clones was inhibited by B9.4 mAb, approximately 15% of these clones were unaffected even in the presence of excess antibody. Flow cytofluorometric analysis of T8 antigen in individual clones did not show any correlation between the amount of T8 antigen expressed, the magnitude of cytolytic activity and the susceptibility (or lack thereof) to inhibition by B9.4 mAb. Of the 16 T4+ CTL clones analyzed, 7 were resistant to inhibition by OKT4 mAb even at doses 10-fold higher than that sufficient for complete inhibition of susceptible clones. Again, no correlation was found between the amount of T4 antigen expressed and the susceptibility to inhibition by the corresponding antibody. The same sets of T8+ and T4+ CTL clones were also analyzed for their susceptibility to inhibition by OKT3 mAb. Although all of the clones expressed the T3 surface antigen, only 15/23 T8+ clones and 9/14 T4+ clones were inhibited by anti-T3 mAb. To further document this clonal heterogeneity, we selected two T3+ T4- T8+ CTL clones that had no concomitant NK-like activity. One clone was resistant to inhibition by OKT3 mAb, whereas the other was highly susceptible. Incubation with OKT3 mAb resulted in modulation of the T3 molecules in both clones. Following modulation, however, the cytolytic activity of the resistant clones was unaffected, whereas the lytic activity of the susceptible clone was abrogated. These results thus indicate extensive clonal heterogeneity in the requirement for T3, T4, and T8 molecules in CTL function. Moreover, it appears that T3 molecules are not always physically and functionally linked to CTL receptor structures.

Human T lymphocyte subpopulations defined by Fc receptors and monoclonal antibodies. A comparison.

Human T cell subpopulations have been defined on the basis of differential expression of either Fc receptors or specific cell-surface antigens. In this study, we utilized a series of monoclonal antibodies reactive with T cells, monocytes, and Ia antigens to characterize isolated subpopulations of T cells bearing receptors for the Fc portion of IgG (T gamma) and subpopulations of T cells bearing receptors for the Fc portion of IgM T mu. The results showed that the T mu population contained both inducer (OKT4+) and cytotoxic/suppressor (OKT5+) populations and was similar to the unfractionated T cell population, whereas the T gamma subset contained few T lymphocytes (OKT3+) and was not enriched for either T cell subset defined by these monoclonal antibodies. Rather, the T gamma population was comprised largely of Ia- cells possessing a monocyte antigen (OKM1+). In reciprocal studies, it was found that both isolated OKT4+ and OKT5+ T cell subsets contained few T gamma cells, whereas both subsets were mainly comprised of T mu cells. We conclude that there is little correlation between T cell subsets defined by these monoclonal antibodies and those defined by Fc receptors.

In vitro expansion of CD3/TCR- human thymocyte populations that selectively lack CD3 delta gene expression: a phenotypic and functional analysis.

Highly purified CD1-3-4-8- human thymocytes were obtained by panning techniques combined with cell depletion with antibody-coated magnetic beads. Most of these cells expressed cytoplasmic CD3 antigen, as assessed by mAbs known to react with the CD3 epsilon chain. After culture with low doses of PMA (0.5 ng/ml) and subsequent addition (at 24 h) of recombinant interleukin 2 (rIL-2; 100 U/ml) cells underwent extensive proliferation (40-60-fold of the initial cell input after 2 wk). The majority of the proliferating cells were CD3-TCR-. The remaining cells (5-40%) were represented by CD3+ TCR gamma/delta+ (BB3- A13+) cells. Further removal of CD3+ TCR-gamma/delta+ cells resulted in highly purified CD3- populations that further proliferated in culture with no substantial phenotypic changes. When CD3+ thymocytes were cultured under the same experimental conditions, only CD3+ TCR-alpha/beta+ cells could be detected, thus indicating that PMA did not affect the surface expression of the CD3/TCR complex, but rather induced preferential growth of CD3- thymocytes. Surface marker analysis of cultured CD3- thymocytes showed that they were homogeneously CD7+, whereas low proportions of cells expressed CD2 and CD8 antigens. Among the natural killer (NK) cell markers, CD56 was highly expressed by all cells, whereas CD16, CD57, CD11b, NKH2, and GL183 were absent. Importantly, these cells were different from peripheral NK cells, as 80-95% of them expressed cytoplasmic CD3 antigen. Functional analysis revealed a strong cytolytic activity against both NK-sensitive (K562) and NK-resistant (M14, Daudi) human target cells. In a redirected killing assay against the Fc gamma R+ P815 cells, mAbs specific for triggering molecules including CD3, CD2, and CD16 failed to augment target cell lysis, while a strong cytolytic effect was induced by PHA. In addition, PHA alone or in combination with PMA induced tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) (but not IL-2) production by CD3- thymocytes. Cloning of fresh CD1-3-4-8-thymocytes in the presence of PMA and rIL-2 resulted in CD3-CD56+ clones that displayed a pattern of cytolytic activity and lymphokine production similar to that of the polyclonal populations. Northern blot analysis of transcripts coding for CD3/TCR molecules revealed the presence of CD3 zeta, epsilon, and gamma transcripts, while CD3 delta was undetectable. Mature transcripts for both gamma and delta TCR chains could be detected, whereas no TCR-alpha mRNA and only a truncated (1.0 kb) form of TCR-beta mRNA were revealed.(ABSTRACT TRUNCATED AT 400 WORDS)

Human peripheral blood lymphocytes bearing T cell receptor gamma/delta. Expression of CD8 differentiation antigen correlates with the expression of the 55-kD, C gamma 2-encoded gamma chain.

We analyzed the CD3-associated molecules present on peripheral blood-derived TCR-gamma/delta+ clones that express CD8 surface antigens. Clones were derived by limiting dilution from CD3+WT31- FACS-purified populations derived from several donors. Eight of greater than 300 TCR-gamma/delta+ clones analyzed expressed CD8 and reacted with delta-TCS-1 mAb. Cell numbers suitable for more detailed analyses could be obtained from four clones, including one derived from thymus. Analysis of CD3-associated TCR molecules immunoprecipitated by anti-Leu-4 (anti-CD3) mAb under conditions that preserve the CD3/TCR association (1% digitonin) showed a predominant 55-60-kD molecule both under reducing and nonreducing conditions. On the other hand, the delta-TCS-1-reactive molecules immunoprecipitated from 25 CD3+ delta-TCS-1+ CD8- clones, in all instances, displayed a 40-44-kD mol mass. In two-dimensional PAGE, TCR-gamma molecules precipitated from delta-TCS-1+ CD8+ clones appeared more acidic than those of BB3+ or delta-TCS-1+ CD8+ clones. Southern analysis confirmed that this type of non-disulphide-linked TCR-gamma/delta is also coded for by the C gamma 2 gene segment.

In vitro proliferation and cloning of CD3- CD16+ cells from human thymocyte precursors.

Purified CD3-4- thymocytes were obtained by depletion of CD3+ and CD4+ cells from fresh thymocyte suspensions. 5-15% of these cells were found to express CD16 antigen, while other natural killer (NK) cell markers were virtually absent. Double fluorescence analysis revealed that 20-40% of thymic CD16+ cells coexpressed CD1, while approximately half were cyCD3+. When cultured in the presence of peripheral blood lymphocytes and H9 leukemia cell line as a source of irradiated feeder cells and interleukin 2 (IL-2), CD3-4- thymocytes underwent extensive proliferation. In addition, after 1-2 wk of culture, 30-50% of these cells were found to express CD16 surface antigen. Cloning under limiting dilution conditions of either CD3-4- or CD3-4-16- thymocytes in the presence of irradiated H9 cells resulted in large proportions (approximately 50%) of CD16+ clones. On the basis of the expression of surface CD16 and/or cyCD3 antigen, clones could be grouped in the following subsets: CD16+ cyCD3+; CD16+ cyCD3-; CD16- cyCD3+; and CD16- cyCD3-. All clones expressed CD56 surface antigen, displayed a strong cytolytic activity against NK sensitive (K562) and NK-resistant (M14) target cells, and produced IFN-gamma and tumor necrosis factor, but not IL-2. Similar to peripheral NK cells, thymic CD16+ cells expressed transcripts for CD16 and for CD3 epsilon (Biassoni, R., S. Ferrini, I. Prigione, A. Moretta, and E.O. Long, 1988. J. Immunol. 140:1685.) and zeta chains (Anderson, P., M. Caligiuri, J. Ritz, and S.F. Schlossman. 1989. Nature [Lond.]. 341:159). Therefore, it appears that cells that are phenotypically and functionally similar to CD3- CD16+ NK cells may arise from immature thymocytes.

Two subsets of human T lymphocytes expressing gamma/delta antigen receptor are identifiable by monoclonal antibodies directed to two distinct molecular forms of the receptor.

Two mAbs directed to the TCR-gamma/delta were analyzed for their pattern of reactivity with CD3+WT31- cell populations or clones. In normal individuals, the BB3 mAb reacted with approximately 2/3 of peripheral blood CD3+WT31- lymphocytes, whereas delta-TCS-1 stained approximately 1/3 of such cells. In addition, the sum of the percentages of BB3+ and delta-TCS-1+ cells approximated the percentages of peripheral blood CD3+WT31- lymphocytes in seven normal donors tested. Also, in peripheral blood-derived polyclonal CD3+WT31- populations, cultured in IL-2, cells reacting with one or another mAb accounted for the whole cell population. On the other hand, only delta-TCS-1-reactive cells, but not BB3+ cells, could be detected in unfractionated as well as in CD4-8-thymocyte populations. Analysis of peripheral blood-derived CD3+WT31- clones showed that 70% of 72 clones analyzed reacted with BB3 mAb, but not with delta-TCS-1 mAb. On the other hand, delta-TCS-1 mAb stained the remaining BB3- clones. Five clones expressing medium-low amounts of CD8 antigen were BB3- delta-TCS-1+. Both types of clones lysed the Fc gamma receptor-bearing P815 target cell in the presence of anti-CD3 mAb (but not of mAb directed against HLA-DR, CD7 molecules, or TCR-alpha/beta). In this cytolytic assay, BB3 mAb induced target cell lysis only by BB3+ clones, whereas delta-TCS-1 mAb was effective only with delta-TCS-1+ clones. The CD3-associated surface molecules expressed by BB3+ or delta-TCS-1+ clones were analyzed after cell surface iodination and immunoprecipitation with the corresponding anti-TCR mAb or with anti-CD3 mAb (in digitonin-containing buffer). In SDS-PAGE, molecules immunoprecipitated from 13 BB3+ clones displayed, under nonreducing conditions, a molecular weight of 80 kD (in some cases, a minor 38-kD band could be detected). Under reducing conditions, two major components of 44 and 41 kD (and a minor component of 38 kD) were detected. On the other hand, TCR molecules immunoprecipitated from 11 different delta-TCS-1+ clones appeared as a diffuse band of 41-44 kD, both under reducing and nonreducing conditions (under non-reducing condition, an additional 38-kD band was present). Therefore, BB3+ cells express a disulphide-linked form of TCR-gamma/delta whereas delta-TCS-1+ cells express a non-disulphide-linked form.(ABSTRACT TRUNCATED AT 400 WORDS)

Morphological and histochemical analyses of two human T-cell subpopulations bearing receptors for IgM or IgG.

Two subpopulation of circulating human T cells forming rosettes with neuraminidase-treated sheep erythrocytes were purified on the basis of the presence of receptors for IgG (TG cells) or for IgM (TM cells), and were shown to have distinguishing morphological and histochemical characteristics. TM cells had the general features of typical small- or medium-sized lymphocytes; most were easily identifiable by distinctive cytoplasmic accumulations, usually one and sometimes two large spots, of nonspecific acid esterase activity. The release of the vesicular contents on short-term culture of TG cells was inhibited by cytochalasin B. Definition of these distinguishing characteristics of TM and TG cells provides a basis for practical enumeration of these functionally distinct subpopulations of human T cells. Some of the TG cells were capable of endocytosis of IgG antibody-coated erythrocytes.

Phenotypic characterization of human cytolytic T lymphocytes in mixed lymphocyte culture.

Mixed lymphocyte reaction (MLR)-activated T cells were analyzed according to the expression of various cell surface markers by the specific cytotoxic T lymphocytes (CTL) generated in the MLR. CTL were found exclusively in a population of MLR-activated T cells that lacked detectable Fc gamma R but that expressed a surface antigen recognized by the 4F2 monoclonal antibody. In contrast, CTL were found in both the Ia-positive and Ia-negative cells after MLR activation. Thus, the specific CTL generated in the allogeneic MLR can be identified and isolated by virtue of the expression of a particular cell surface marker.

Natural killer alloeffector responses in haploidentical hemopoietic stem cell transplantation to treat high-risk leukemias.

Natural killer (NK) cells, a major cell type of the innate immunity, express surface receptors that regulate potent effector functions such as cytolytic activity and release of cytokines playing a central role in inflammatory response and immunoregulation. In this contribution, we briefly outline the major steps from the discovery of human leukocyte antigen (HLA)-class I-specific inhibitory receptors in humans to recent successful clinical applications in the cure of high-risk leukemias both in adults and in pediatric patients. A central role is played by 'alloreactive' NK cells originated from donor's CD 34(+) cells in eradicating leukemic cells in the setting of T-cell-depleted haploidentical hemopoietic stem cell transplantation. Because alloreactive NK cells play a central role also in preventing graft rejection and graft-vs-host disease, they may represent an ideal tool to treat patients affected by acute high-risk leukemias.

Inhibitory checkpoints in human natural killer cells: IUPHAR Review 28.

Immune checkpoint inhibitors have revolutionized cancer therapy leading to exceptional success. However, there is still the need to improve their efficacy in non-responder patients. Natural killer (NK) cells represent the first line of defence against tumours, due to their ability to release immunomodulatory cytokines and kill target cells that have undergone malignant transformation. Harnessing NK cell response will open new possibilities to improve control of tumour growth. In this respect inhibitory checkpoints expressed on these innate lymphocytes represents a promising target for next-generation immunotherapy. In this review, we will summarize recent evidences on the expression of NK cells receptors in cancer, with a focus on the inhibitory checkpoint programmed cell death protein 1 (PD-1). We will also highlight the strength and limitations of the blockade of PD-1 inhibitory pathway and suggest new combination strategies that may help to unleash more efficiently NK cell anti-tumour response.

Internalization via Antennapedia protein transduction domain of an scFv antibody toward c-Myc protein.

We constructed a single-chain variable fragment miniantibody (G11-scFv) directed toward the transactivation domain of c-Myc, which is fused with the internalization domain Int of Antennapedia at its carboxyl terminus (a cargo-carrier construct). In ELISA experiments, an EC(50) for binding saturation was achieved at concentrations of G11-scFv-Int(-) of approximately 10(-8) M. Internalization of a fluoresceinated Fl-G11-scFv-Int(+) construct was observed in intact human cultured cells with confocal microscopy. After 5 h of incubation in medium containing 1 microM Fl-G11-scFv-Int(+) or Fl-G11-scFv-Int(-), fluorescence intensity was determined in individual cells, both for cytoplasmic and nuclear compartments: concentration levels of Fl-G11-scFv-Int(+), relative to the extracellular culture medium concentration, were 4-5 times higher in the cytoplasm, 7-8 times higher in the nucleus, and 10 times higher in the nucleoli. In the same experimental conditions, the Fl-G11-scFv-Int(-) construct was 3-4 times more concentrated outside of the cells than inside. Cell membranes kept their integrity after 5 h of incubation. The antiproliferative activity of our miniantibody was studied on HCT116 cells. Incubation with 4 microM G11-scFv-Int(+) for 4 days induced very significant statistical and biological growth inhibition, whereas Int alone was completely inactive. Miniantibodies capable of penetrating cell membranes dramatically broaden the potential for innovative therapeutic agents and attack of new targets.

4F2 monoclonal antibody recognizes a surface antigen on spread human fibroblasts of embryonic but not of adult origin.

The 4F2 monoclonal antibody (mAb) has been shown to recognize a 120-kilodalton glycoprotein expressed on the cell surface of human peripheral blood monocytes, activated (but not resting) T or B cells, and T and B lymphoblastoid cell lines. In this report we show that 4F2 mAb specifically binds to the surface of adherent human embryonic fibroblasts but fails to bind to normal adult fibroblasts. Moreover, 4F2 antigen was expressed on sarcoma-derived or SV40-transformed adult fibroblastic cells. Finally, addition of 4F2 mAb inhibited the growth of cultured HT-1080 fibrosarcoma cell line, but had no inhibitory effect on various embryonic and adult normal or transformed fibroblasts.

Purification and HPLC-MS analysis of a naturally processed HCMV-derived peptide isolated from the HEK-293T/HLA-E+/Ul40+ cell transfectants and presented at the cell surface in the context of HLA-E.

A new method for isolation and characterization of peptides presented in the context of the nonclassical human leukocytes antigen (HLA) class I molecule HLA-E was developed. A combination of different chromatographic steps coupled with electrospray mass spectrometry allowed us to detect the presence of small amounts of a naturally processed human Cytomegalovirus (HCMV)-derived peptide isolated from the HEK-293T/HLA-E+/UL40+ transfected cells of from HELA cell line. The peptide sequence was confirmed by tandem mass spectrometry (MS/MS). This approach provides a versatile and sensitive method for direct identification of MHC class I-binding peptides that might be derive from different pathogen or tumor-associated proteins.

IL15 induces a potent antitumor activity in NK cells isolated from malignant pleural effusions and overcomes the inhibitory effect of pleural fluid.

Natural Killer (NK) cells are capable of recognizing and killing cancer cells and play an important role in tumor immunosurveillance. However, tumor-infiltrating NK cells are frequently impaired in their functional capability. A remarkable exception is represented by NK cells isolated from malignant pleural effusions (PE) that are not anergic and, upon IL2-induced activation, efficiently kill tumor cells. Although IL2 is used in various clinical trials, severe side effects may occur in treated patients. In this study, we investigated whether also other clinical-grade cytokines could induce strong cytotoxicity in NK cells isolated from pleural fluid of patients with primary or metastatic tumors of different origins. We show that PE-NK cells, cultured for short-time intervals with IL15, maintain the CD56bright phenotype, a high expression of the main activating receptors, produce cytokines and kill tumor cells in vitro similarly to those treated with IL2. Moreover, IL15-activated PE-NK cells could greatly reduce the growth of established tumors in mice. This in vivo antitumor effect correlated with the ability of IL15-activated PE-NK cells to traffic from periphery to the tumor site. Finally, we show that IL15 can counteract the inhibitory effect of the tumor pleural microenvironment. Our study suggests that IL15-activated NK cells isolated from pleural fluid (otherwise discarded after thoracentesis) may represent a suitable source of effector cells to be used in adoptive immunotherapy of cancer.

Group 3 innate lymphoid cells regulate neutrophil migration and function in human decidua.

Innate lymphoid cells (ILCs) have a central role in innate defenses against pathogens, lymphoid organogenesis, and tissue remodeling. They have been detected in human decidua, however, their role in this tissue remains unclear. Successful pregnancy requires an early inflammatory phase favoring implantation and tissue remodeling as well as a subsequent regulatory phase to prevent fetal rejection and supporting neoangiogenesis. Here, we show that, during the first trimester of pregnancy, neutrophils infiltrate decidua basalis and are more abundant in normal pregnancy than in spontaneous miscarriages. Decidual neutrophils localize in proximity of NCR+ILC3, which may influence neutrophil migration and survival given their production of CXCL8 and granulocyte macrophage colony-stimulating factor (GM-CSF). Moreover, NCR+ILC3-derived GM-CSF was found to induce the expression of heparin-binding EGF-like growth factor and IL1ra in neutrophils, two proteins/cytokines involved in tissue remodeling and maintenance of pregnancy. Our data suggest that the simultaneous presence of NCR+ILC3 and neutrophils in decidual tissues and their possible cross talk, may have a role in the early phases of pregnancy.

Human natural killer cell function and receptors.

Recent years have witnessed major progress in our understanding of the molecular mechanisms regulating natural killer cell (NK cell) function. These advances stem primarily from the discovery of a number of receptors specific for major histocompatibility complex (MHC) class I and, more recently, of the activating receptors and coreceptors responsible for natural cytotoxicity. Important studies performed over the past year have allowed us to define the evolution of the MHC-specific inhibitory receptors by comparative analysis in different species. The roles of the 'activating natural cytotoxicity receptors', NKG2D and certain coreceptors in the lysis of different tumors have been defined in detail. The mechanism by which the 2B4 coreceptor renders patients with X-linked lymphoproliferative disease unable to control Epstein-Barr virus has been elucidated. Inhibitory receptors identified in NK cells may also be expressed by normal and leukemic myeloid cells, in which they can block cell proliferation and survival. It has also become clear that viruses such as cytomegalovirus have evolved strategies to interfere with NK-cell function to protect themselves from NK-mediated attack.