Unique subpopulations of CD56+ NK and NK-T peripheral blood lymphocytes identified by chemokine receptor expression repertoire

CD56, an adhesion molecule closely related to neural cell adhesion molecule, is an immunophenotypic marker for several unique populations of PBLS: Although CD56(+) cells derive from multiple lymphocyte lineages, they share a role in immunosurveillance and antitumor responses. We have studied the chemokine receptor expression patterns and functional migratory responses of three distinct CD56(+) populations from human peripheral blood. NK-T cells were found to differ greatly from NK cells, and CD16(+) NK cells from CD16(-) NK cells. CD16(+) NK cells were the predominant population responding to IL-8 and fractalkine, whereas NK-T cells were the predominant population responding to the CCR5 ligand macrophage-inflammatory protein-1beta. CD16(-) NK cells were the only CD56(+) population that uniformly expressed trafficking molecules necessary for homing into secondary lymphoid organs through high endothelial venule. These findings describe a diverse population of cells that may have trafficking patterns entirely different from each other, and from other lymphocyte types.     

An indirect role for NK cells in a CD4(+) T-cell-dependent mouse model of type I diabetes

CD8(+) T cells kill pancreatic β-cells in a cell-cell contact-dependent mechanism in the non-obese diabetic mouse. CD4(+) T lymphocytes are also able to kill pancreatic β-cells, but they do not directly contact β-cells and may use another cell type as the actual cytotoxic cell. Natural killer (NK) cells could have this role but it is uncertain whether they are cytotoxic towards β-cells. Therefore, the requirement for NK cells in β-cell destruction in the CD4-dependent T-cell antigen receptor transgenic NOD4.1 mice was examined. NK cells failed to kill β-cells in vitro, even in the absence of major histocompatibility complex class I. We observed only 9.7±1.1% of islet infiltrating NK cells from NOD4.1 mice expressing the degranulation marker CD107a. Diabetogenic CD4(+) T cells transferred disease to NODscid.IL2Rγ(-/-) mice lacking NK cells, indicating that NK cells do not contribute to β-cell death in vitro or in vivo. However, depletion of NK cells reduced diabetes incidence in NOD4.1 mice, suggesting that NK cells may help to maintain the right environment for cytotoxicity of effector cells.     

IL-15 expands unconventional CD8alphaalphaNK1.1+ T cells but not Valpha14Jalpha18+ NKT cells

Despite recent gains in knowledge regarding CD1d-restricted NKT cells, very little is understood of non-CD1d-restricted NKT cells such as CD8(+)NK1.1(+) T cells, in part because of the very small proportion of these cells in the periphery. In this study we took advantage of the high number of CD8(+)NK1.1(+) T cells in IL-15-transgenic mice to characterize this T cell population. In the IL-15-transgenic mice, the absolute number of CD1d-tetramer(+) NKT cells did not increase, although IL-15 has been shown to play a critical role in the development and expansion of these cells. The CD8(+)NK1.1(+) T cells in the IL-15-transgenic mice did not react with CD1d-tetramer. Approximately 50% of CD8(+)NK1.1(+) T cells were CD8alphaalpha. In contrast to CD4(+)NK1.1(+) T cells, which were mostly CD1d-restricted NKT cells and of which approximately 70% were CD69(+)CD44(+), approximately 70% of CD8(+)NK1.1(+) T cells were CD69(-)CD44(+). We could also expand similar CD8alphaalphaNK1.1(+) T cells but not CD4(+) NKT cells from CD8alpha(+)beta(-) bone marrow cells cultured ex vivo with IL-15. These results indicate that the increased CD8alphaalphaNK1.1(+) T cells are not activated conventional CD8(+) T cells and do not arise from conventional CD8alphabeta precursors. CD8alphaalphaNK1.1(+) T cells produced very large amounts of IFN-gamma and degranulated upon TCR activation. These results suggest that high levels of IL-15 induce expansion or differentiation of a novel NK1.1(+) T cell subset, CD8alphaalphaNK1.1(+) T cells, and that IL-15-transgenic mice may be a useful resource for studying the functional relevance of CD8(+)NK1.1(+) T cells.     

CD56 identifies monocytes and not natural killer cells in rhesus macaques.

BACKGROUND: CD56 is a lineage-specific marker of human natural killer (NK) cells. There are conflicts in the literature regarding the role of CD56 as a marker of NK cells in non-human primates. In the present study, we examined the role of CD56 in identifying rhesus NK cells. METHODS: The immunophenotype of normal macaque and human NK cells was analyzed by two- and three-color flow cytometry. Flow cytometric cell sorting was subsequently used to deplete or purify NK cells; the resulting cell populations were then used in standard chromium release assays of NK lytic function. RESULTS: In peripheral blood mononuclear cells of the rhesus macaque, CD56 was expressed primarily on cells with the light scatter and immunophenotypic profile of monocytes. Flow cytometric depletion of rhesus CD56(+) monocytic cells did not diminish functional activity against K562 cells, whereas depletion of CD8(+) or CD16(+) lymphocytes completely abrogated functional activity. Three-color flow cytometric analysis of CD8(+), CD16(+) lymphocytes showed that they expressed other markers (CD2, CD7, TIA-1) associated with NK cells, but notably, not CD56. CONCLUSIONS: These studies demonstrate that CD56 is not suitable as a marker of NK cells in the rhesus macaque.     

Phenotypic characteristics of natural killer cells in acute hepatitis

Natural killer (NK) cells are the principal effector cell population in innate immune defense against many types of infections. These cells are enriched in the liver, where they comprise approximately 40% to 60% of the intrahepatic lymphocyte pool compared to the peripheral blood compartment. In chronic HBV and HCV infection, NK cells were reported to be partially dysfunctional due to impaired IFN-Γ secretion. Few studies have examined phenotypic features of NK cells in acute hepatitis. We identified NK (CD56+CD3-) cell populations in patients with acute hepatitis by examining the expressions of phenotypic NK cell markers (CD16, NKG2A, and NKG2D). Peripheral blood mononuclear cells were isolated from patients with acute hepatitis A (7) and patients with non-viral acute toxic hepatitis (6) during the symptomatic and convalescent phases. Expressions of NK (CD56+CD3-) cell markers, CD16, NKG2A, and NKG2D, were measured by flow cytometry. Symptomatic acute hepatitis including non-viral hepatitis and HAV infection showed significant increases of NKG2A expression compared to healthy controls. Interestingly, there was a direct correlation between the proportion of NK cell populations and liver function parameters (AST, ALT) in HAV infection. The strong correlation was also observed between the expression of NKG2A+NK cells and ALT, which suggests that most of NK cells in severe phase of disease express high level of NKG2A on their surface. In addition, decreased number of NK cells (CD56+CD3-) in symptomatic phase began to increase in the convalescent phase of acute hepatitis A. However, the expression of NKG2A tended to be reduced, which indicates that NKG2A, the inhibitory receptor on NK cells, can be a severity parameter in acute hepatitis.     

Turnover rates of B cells,T cells,and NK cells in simian immunodeficiency virus-infected and uninfected rhesus macaques

We determined average cellular turnover rates by fitting mathematical models to 5-bromo-2'-deoxyuridine measurements in SIV-infected and uninfected rhesus macaques. The daily turnover rates of CD4(+) T cells, CD4(-) T cells, CD20(+) B cells, and CD16(+) NK cells in normal uninfected rhesus macaques were 1, 1, 2, and 2%, respectively. Daily turnover rates of CD45RA(-) memory T cells were 1%, and those of CD45RA(+) naive T cells were 0.5% for CD4(+) T cells and approximately 1% for CD4(-)CD45RA(+) T cells. In SIV-infected monkeys with high viral loads, the turnover rates of T cells were increased approximately 2-fold, and that of memory T cells approximately 3-fold. The turnover of CD4(+)CD45RA(+) naive T cells was increased 2-fold, whereas that of CD4(-)CD45RA(+) naive T cells was marginally increased. B cells and NK cells also had increased turnover in SIV-infected macaques, averaging 3 and 2.5% per day, respectively. For all cell types studied here the daily turnover rate increased with the decrease of the CD4 count that accompanied SIV infection. As a consequence, the turnover rates of CD4(+) T cells, CD4(-) T cells, B cells, and NK cells within each monkey are strongly correlated. This suggests that the cellular turnover of different lymphocyte populations is governed by a similar process which one could summarize as "generalized immune activation." Because the viral load and the CD4 T cell count are negatively correlated we cannot determine which of the two plays the most important role in this generalized immune activation.     

Therapeutic efficacy of TBC3711 in monocrotaline-induced pulmonary hypertension

Background

Modified function of immune cells in nasal secretions may play a role in the enhanced susceptibility to respiratory viruses that is seen in smokers. Innate immune cells in nasal secretions have largely been characterized by cellular differentials using morphologic criteria alone, which have successfully identified neutrophils as a significant cell population within nasal lavage fluid (NLF) cells. However, flow cytometry may be a superior method to fully characterize NLF immune cells. We therefore characterized immune cells in NLF by flow cytometry, determined the effects of live attenuated influenza virus (LAIV) on NLF and peripheral blood immune cells, and compared responses in samples obtained from smokers and nonsmokers.

Methods

In a prospective observational study, we characterized immune cells in NLF of nonsmokers at baseline using flow cytometry and immunohistochemistry. Nonsmokers and smokers were inoculated with LAIV on day 0 and serial nasal lavages were collected on days 1-4 and day 9 post-LAIV. LAIV-induced changes of NLF cells were characterized using flow cytometry. Cell-free NLF was analyzed for immune mediators by bioassay. Peripheral blood natural killer (NK) cells from nonsmokers and smokers at baseline were stimulated in vitro with LAIV followed by flow cytometric and mediator analyses.

Results

CD45(+)CD56(-)CD16(+) neutrophils and CD45(+)CD56(+) NK cells comprised median 4.62% (range 0.33-14.52) and 23.27% (18.29-33.97), respectively, of non-squamous NLF cells in nonsmokers at baseline. LAIV did not induce changes in total NK cell or neutrophil percentages in either nonsmokers or smokers. Following LAIV inoculation, CD16(+) NK cell percentages and granzyme B levels increased in nonsmokers, and these effects were suppressed in smokers. LAIV inoculation enhanced expression of activating receptor NKG2D and chemokine receptor CXCR3 on peripheral blood NK cells from both nonsmokers and smokers in vitro but did not induce changes in CD16(+) NK cells or granzyme B activity in either group.

Conclusions

These data are the first to identify NK cells as a major immune cell type in the NLF cell population and demonstrate that mucosal NK cell cytotoxic function is suppressed in smokers following LAIV. Altered NK cell function in smokers suggests a potential mechanism that may enhance susceptibility to respiratory viruses.     

Nasal lavage natural killer cell function is suppressed in smokers after live attenuated influenza virus

Background

Modified function of immune cells in nasal secretions may play a role in the enhanced susceptibility to respiratory viruses that is seen in smokers. Innate immune cells in nasal secretions have largely been characterized by cellular differentials using morphologic criteria alone, which have successfully identified neutrophils as a significant cell population within nasal lavage fluid (NLF) cells. However, flow cytometry may be a superior method to fully characterize NLF immune cells. We therefore characterized immune cells in NLF by flow cytometry, determined the effects of live attenuated influenza virus (LAIV) on NLF and peripheral blood immune cells, and compared responses in samples obtained from smokers and nonsmokers.

Methods

In a prospective observational study, we characterized immune cells in NLF of nonsmokers at baseline using flow cytometry and immunohistochemistry. Nonsmokers and smokers were inoculated with LAIV on day 0 and serial nasal lavages were collected on days 1-4 and day 9 post-LAIV. LAIV-induced changes of NLF cells were characterized using flow cytometry. Cell-free NLF was analyzed for immune mediators by bioassay. Peripheral blood natural killer (NK) cells from nonsmokers and smokers at baseline were stimulated in vitro with LAIV followed by flow cytometric and mediator analyses.

Results

CD45(+)CD56(-)CD16(+) neutrophils and CD45(+)CD56(+) NK cells comprised median 4.62% (range 0.33-14.52) and 23.27% (18.29-33.97), respectively, of non-squamous NLF cells in nonsmokers at baseline. LAIV did not induce changes in total NK cell or neutrophil percentages in either nonsmokers or smokers. Following LAIV inoculation, CD16(+) NK cell percentages and granzyme B levels increased in nonsmokers, and these effects were suppressed in smokers. LAIV inoculation enhanced expression of activating receptor NKG2D and chemokine receptor CXCR3 on peripheral blood NK cells from both nonsmokers and smokers in vitro but did not induce changes in CD16(+) NK cells or granzyme B activity in either group.

Conclusions

These data are the first to identify NK cells as a major immune cell type in the NLF cell population and demonstrate that mucosal NK cell cytotoxic function is suppressed in smokers following LAIV. Altered NK cell function in smokers suggests a potential mechanism that may enhance susceptibility to respiratory viruses.     

Population dynamics of human activated natural killer cells in culture

The growth kinetics and population dynamics of recombinent interleukin-2 (rlL-2) stimulated human natural killer (NK) cell-enriched populations were studied in vitro. The NK-enriched populations was obtained from normal peripheral blood mononuclear cells (PBMNC) by immunomagnetic bead depletion of CD3(+) and CD5(+) T cells. The growth kinetics of NK cells, T cells, monocytes, and total cells are shown. In the absence of PBMNC accessory cells, the NK-enriched population showed limited expansion. In the presence of PBMNC accessory cells, the NK-enriched population expanded threefold more than in the absence of accessory cells due to increased NK cell growth rate and increased duration of exponential growth. Using a Transwell system, which separates two cell population by a polycarbonate membrane, the accessory cells were shown to act on the NK-enriched population via a diffusible factor. Accessory cell conditioned media was able to replace the accessory cell population to stimulate NK cell expansion. A monocyte-enriched population prepared by sheep red blood cell rosetting of T cells was extensively phenotyped and compared with the NK-enriched populations. Although the final cultured cells were phenotypically homogeneous for CD56(+)/CD3(-) NK cells, the initial NK precusor populations appear to be different. Namely, the NK cell precursors in the monocyte-enriched population were predominantly CD56(+)/CD2(-). Kinetic equations were formulated for this culture system and the effects of major culture variables are investigated.     

The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T lymphocytes

We examined the antigenic and functional characteristics of human peripheral blood lymphocytes that differentially express the CD16 (Leu-11) and Leu-19 (NKH-1) antigens. Leu-19 is a approximately 220,000 daltons protein expressed on approximately 15% of freshly isolated peripheral blood lymphocytes. Within the Leu-19+ subset, three distinct populations were identified: CD3-,CD16+,Leu-19+ cells; CD3+,CD16-,Leu-19+ cells; and CD3-,CD16-,Leu-19bright+ cells. Both the CD3+,CD16-,Leu-19+ and CD3-,CD16+,Leu-19+ populations mediated non-major histocompatibility complex (MHC)-restricted cytotoxicity against the NK-sensitive tumor cell K562 and were large granular lymphocytes. CD3-,CD16+,Leu-19+ NK cells were the most abundant (comprising approximately 10% of peripheral blood lymphocytes) and the most efficient cytotoxic effectors. The finding that CD3+,Leu 19+ lymphocytes mediated cytotoxicity against K562 unequivocally demonstrates that a unique subset of non-MHC-restricted cytotoxic CD3+ T lymphocytes are present in the peripheral blood of unprimed, normal individuals. However, CD3+,CD16-,Leu-19+ cells comprised less than 5% of peripheral blood lymphocytes, and the cytotoxic activity of this subset was significantly less than CD3-,CD16+,Leu-19+ NK cells. Most CD3+,Leu-19+ T cells co-expressed the CD2, CD8, and CD5 differentiation antigens. The antigenic and functional phenotype of peripheral blood CD3+,Leu-19+ cytotoxic T lymphocytes corresponds to the interleukin 2-dependent CD3+ cell lines that mediate non-MHC-restricted cytotoxicity against NK-sensitive tumor cell targets. A small population of Leu-19bright+ lymphocytes lacking both CD3 and CD16 was also observed. This population (comprising less than 2% of peripheral blood lymphocytes) contained both large agranular lymphocytes and large granular lymphocytes. CD3-,CD16-,Leu-19bright+ lymphocytes also mediate non-MHC-restricted cytotoxicity. The relationship of these CD3-CD16-,Leu-19bright+ lymphocytes to CD3+ T cells or CD16+ NK cells is unknown.     

Migration and maturation of human colonic dendritic cells

Dendritic cells (DC) in the colon may regulate intestinal immunity but remain poorly characterized. In this study a CD11c(+)HLA-DR(+)lin(-) (CD3(-)CD14(-)CD16(-)CD19(-)CD34(-)) population has been identified by flow cytometry in cells obtained by rapid collagenase digestion of human colonic and rectal biopsies. These day 0 (d0) CD11c(+)HLA-DR(+)lin(-) cells comprised approximately 0.6% of the mononuclear cells obtained from the lamina propria, were endocytically active, and had the phenotype of immature DC; they were CD40(+) and expressed low levels of CD83 and CD86, but little or no CD80 or CD25. Similar d0 DC populations were isolated from the colonic mucosa of healthy controls and from both inflamed and noninflamed tissue from patients with Crohn's disease. The lamina propria also contained a population of cells capable of migrating out of biopsies during an overnight culture and differentiating into mature DC with lower levels of endocytic activity and high cell surface expression of CD40, CD80, CD86, CD83, and CD25. This mature DC population was a potent stimulator of an allogeneic mixed leukocyte (MLR). Overnight culture of cells isolated by enzymatic digestion on d0 yielded DC with a phenotype intermediate between that of the d0 cells and that of the cells migrating out overnight. Overnight culture of colonic cells in which DC and HLA-DR(+)lin(+) cells were differentially labeled with FITC-dextran suggested that some of the maturing DC might differentiate from HLA-DR(+)lin(+) progenitors. This study presents the first analysis of the phenotype, maturational status, and migratory activity of human gut DC.     

The effect of interleukin-15 on the expression of killer-cell immunoglobulin-like receptors on peripheral natural killer cells in human

Interleukin (IL)-15 has emerged as a key regulator of both natural killer (NK) cell differentiation and activation. The aim of the present study was to investigate the expansion of the population of cells expressing killer-cell immunoglobulin-like receptors (CD158a and CD158b) in human peripheral lymphocytes by treatment with IL-15. One million peripheral lymphocytes were cultured in RPMI1640 medium alone or in medium containing IL-2 at 100 U/ml or IL-15 at 0.1, 1.0, or 10.0 ng/ml for 48 h. After each incubation, we assessed the natural killing activity and the population of CD16(+)CD158a(+)/b(+) cells and CD8(+)CD158a(+)/b(+) cells. IL-15 increased the NK activity and expanded the populations of CD16(+)CD158a(+)/b(+) cells and CD8(+)CD158a(+)/b(+) cells. These actions were dose dependent, and the effects of IL-15 at 1.0 ng/ml were close to those of IL-2 at 100 U/ml. These findings suggest that IL-15 induces the effector functions of resting NK cells throughout the body, and thereby plays a critical role in the activation of tissue-associated immune responses.     

Phylogeny of natural cytotoxicity: cytotoxic activity of coelomocytes of the purple sea urchin, Arbacia punctulata.

Coelomocyte-mediated nonspecific cell cytotoxic activity against human and murine target cells by the purple sea urchin Arbacia punctulata was investigated in vitro. Cytotoxic activity toward target cells was shown to be mediated by different coelomocyte populations isolated by discontinuous density gradient centrifugation. The population of phagocytic amebocytes showed the strongest cytotoxic activity and the highest binding to human NK markers by cytometry analysis. Our immunophenotypic studies showed that A. punctulata phagocytic amebocytes are CD14(+), CD56(+), CD158b(+), CD3(-), CD4(-), CD8(-), and CD16(-). The cytotoxic activity was independent of experimental incubation temperatures, required viable effector cells, and required cell-cell contact between the effector and target cells. Sodium azide significantly decreased coelomocyte cytotoxicity, indicating that cytotoxicity is metabolically dependent, and EDTA reduction of cytotoxic activity is consistent with the involvement of divalent cations in the cytotoxic process. These data describe a population of sea urchin coelomocytes (the phagocytic amebocyte) that are CD14(+), CD56(+), and CD158b(+), with cytotoxic activities.     

A major fraction of human bone marrow lymphocytes are Th2-like CD1d-reactive T cells that can suppress mixed lymphocyte responses.

Murine bone marrow (BM) NK T cells can suppress graft-vs-host disease, transplant rejection, and MLRs. Human BM contains T cells with similar potential. Human BM was enriched for NK T cells, approximately 50% of which recognized the nonpolymorphic CD1d molecule. In contrast to the well-characterized blood-derived CD1d-reactive invariant NK T cells, the majority of human BM CD1d-reactive T cells used diverse TCR. Healthy donor invariant NK T cells rapidly produce large amounts of IL-4 and IFN-gamma and can influence Th1/Th2 decision-making. Healthy donor BM CD1d-reactive T cells were Th2-biased and suppressed MLR and, unlike the former, responded preferentially to CD1d(+) lymphoid cells. These results identify a novel population of human T cells which may contribute to B cell development and/or maintain Th2 bias against autoimmune T cell responses against new B cell Ag receptors. Distinct CD1d-reactive T cell populations have the potential to suppress graft-vs-host disease and stimulate antitumor responses.     

Distribution of Several Activating and Inhibitory Receptors on CD3<Superscript>−</Superscript>CD16<Superscript>+</Superscript> NK Cells and Their Correlation with NK Cell Function in Healthy Individuals

The aim of this study was to estimate the distribution and density of a representative set of activating and inhibitory receptors on gated natural killer (NK) cells, as well as on their bright and dim subsets, and to correlate the receptor expression with NK cell activity for healthy individuals on CD3⁻CD16⁺ NK cells. We show that in 43 healthy controls NK cell activity against K562 target cells was 37.34% (E:T, 80:1) by standard chromium release assay. The expression of receptors on NK cells and their subsets was analyzed by flow cytometry. The cytotoxic CD3⁻CD16^bright NK subset constituted 78.97%, while the regulatory CD3⁻CD16^dim NK subset constituted 21.03% of NK cells. We show the distribution of NKG2D, CD161, CD158a, and CD158b receptors on CD3⁻CD16⁺ NK cells in peripheral blood lymphocytes (PBLs), on gated NK cells, and on the CD3⁻CD16^bright and CD3⁻CD16^dim subsets. Contrary to CD158a and CD158b killer immunoglobulin-like receptors (KIRs), there is a significant positive correlation of NKG2D and CD161 expression with NK cytotoxicity. We show the kinetics of change in CD3⁻CD16⁺NK/K562 conjugate composition, together with the stronger target binding capacity of CD16^bright NK cells. Furthermore, we show that after coculture of PBLs with K562 the expression of CD107a, a degranulation marker, on CD3⁻CD16⁺NK cells and subsets is time dependent and significantly higher on the cytotoxic CD3⁻CD16^bright NK subset. The novel data obtained regarding expression of NK cell activating and inhibitory receptors for healthy individuals may aid in detecting changes that are associated with various diseases.     

Memory CD8+ T cells provide an early source of IFN-gamma

During the non-Ag-specific early phase of infection, IFN-gamma is believed to be primarily provided by NK and NKT cells in response to pathogen-derived inflammatory mediators. To test whether other cell types were involved in early IFN-gamma release, IFN-gamma-producing cells were visualized in spleens and lymph nodes of LPS-injected mice. In addition to NK and NKT cells, IFN-gamma was also detected in a significant fraction of CD8(+) T cells. CD8(+) T cells represented the second major population of IFN-gamma-producing cells in the spleen ( approximately 30%) and the majority of IFN-gamma(+) cells in the lymph nodes ( approximately 70%). LPS-induced IFN-gamma production by CD8(+) T cells was MHC class I independent and was restricted to CD44(high) (memory phenotype) cells. Experiments performed with C3H/HeJ (LPS-nonresponder) mice suggested that CD8(+) T cells responded to LPS indirectly through macrophage/dendritic cell-derived IFN-alpha/beta, IL-12, and IL-18. IFN-gamma was also detected in memory CD8(+) T cells from mice injected with type I IFN or with poly(I:C), a synthetic dsRNA that mimics early activation by RNA viruses. Taken together, these results suggest that in response to bacterial and viral products, memory T cells may contribute to innate immunity by providing an early non-Ag-specific source of IFN-gamma.     

Visualization and characterization of respiratory syncytial virus F-specific CD8(+) T cells during experimental virus infection

CTL play a major role in the clearance of respiratory syncytial virus (RSV) during experimental pulmonary infection. The fusion (F) glycoprotein of RSV is a protective Ag that elicits CTL and Ab response against RSV infection in BALB/c mice. We used the strategy of screening a panel of overlapping synthetic peptides corresponding to the RSV F protein and identified an immunodominant H-2K(d)-restricted epitope (F(85-93); KYKNAVTEL) recognized by CD8(+) T cells from BALB/c mice. We enumerated the F-specific CD8(+) T cell response in the lungs of infected mice by flow cytometry using tetramer staining and intracellular cytokine synthesis. During primary infection, F(85-93)-specific effector CD8(+) T cells constitute approximately 4.8% of pulmonary CD8(+) T cells at the peak of the primary response (day 8), whereas matrix 2-specific CD8(+) T cells constituted approximately 50% of the responding CD8(+) T cell population in the lungs. When RSV F-immune mice undergo a challenge RSV infection, the F-specific CD8(+) T cell response is accelerated and dominates, whereas the primary response to the matrix 2 epitope in the lungs is reduced by approximately 20-fold. In addition, we found that activated F-specific effector CD8(+) T cells isolated from the lungs of RSV-infected mice exhibited a lower than expected frequency of IFN-gamma-producing CD8(+) T cells and were significantly impaired in ex vivo cytolytic activity compared with competent F-specific effector CD8(+) T cells generated in vitro. The significance of these results for the regulation of the CD8(+) T cell response to RSV is discussed.     

Differential production of IFN-gamma, analyzed at the single-cell level, by specific subsets of human NK and T cells from healthy and HIV(+) subjects

BACKGROUND: Interferon gamma is a cytokine that plays a central role in immunity, and is physiologically secreted by T and NK cells under appropriate stimuli during the immune response. By means of flow cytometry, we performed a single cell analysis of interferon gamma producing NK cells and their surface phenotype in normal and HIV(+) individuals that show several defects of cytokine production and cellular immunity. METHODS: PBMC or purified NK cells were stimulated for 1-12 h with PMA/ionomycin in the presence of monensin, subsequently stained for surface CD56 and CD3 or CD8, and for intracytoplasmic IFN-gamma, and analysed by flow cytometry. RESULTS: Our results show that CD56(+) NK cells are more efficient interferon gamma producers than T cells. Moreover, within the CD56(+) NK cell population, those that co-express low density CD8 are the best producers. Finally, we show that NK cells during HIV infection are more massively recruited to interferon gamma production than those from normal subjects. CONCLUSIONS: Both in the normal and HIV(+) subjects, a higher percentage of NK cells than T cells can produce IFN-gamma although differences can be identified within the NK cells subset in terms of IFN-gamma production. The production of IFN-gamma is fully achievable in the HIV(+) subjects, which is consistent with their elevated plasmatic levels of the cytokine. The possibility that NK cells that produce interferon gamma could represent a functionally distinct population committed to the production of this cytokine, is discussed.     

Preparation of spermatogenic cell populations at specific stages of differentiation in the human.

Studying biochemical events in human spermatogenesis requires separated populations of spermatogenic cells. Dissociation of these cells was performed by a Trypsin-DNAse method adapted from the technique used for rodents. Cell separation was performed by centrifugal elutriation. Seven populations were collected, one further purified by Percoll gradient centrifugation, giving nine different cell populations. The efficiency of the cell separation was evaluated by phase contrast microscopy, flow cytometric DNA analysis, and electron microscopy. Five populations were enriched in spermatids: two in round spermatids (87% and 73%), another in round (52%) and elongating (44%) spermatids, another constituted by 80% elongating spermatids, and the last by 90% elongated spermatids. Two of the four remaining populations were enriched in primary spermatocytes (74% and 54%); another population was the upper part of the Percoll gradient and constituted cytoplasmic lobes and residual bodies (89%); the last population was made up of various cells, with no specific enrichment. Electron microscopic observations revealed good preservation of the separated cells; only the flagella from elongated spermatids were lost. Furthermore, an unusual pattern of nucleoplasm distribution during stages 2-4 of spermatid differentiation was observed and its signification is discussed with regard to the shape of the human spermatozoon.