Elevated gamma interferon-producing NK cells, CD45RO memory-like T cells, and CD4 T cells are associated with protection against malaria infection in pregnancy

Previous studies have shown that gamma interferon (IFN-gamma) production in the placenta is associated with protection against placental malaria. However, it remains unknown which IFN-gamma-producing cell subpopulations are involved in this protection and whether the cellular immune components of protection are the same in the peripheral and the placental blood compartments. We investigated cell subpopulations for CD4, CD8, and CD45RO memory-like T cells and CD56+/CD3- natural killer (NK) cells and for IFN-gamma production by these cells in maternal peripheral and placental intervillous blood in relation to the status of malaria infection in pregnancy. Of 52 human immunodeficiency virus-negative enrolled pregnant women residing in Western Kenya, 20 had placental parasitemia. We found that the percentages of CD45RO memory-like and CD4 T cells were significantly higher in the periphery than in the placenta, while the CD56/CD3- NK-cell percentage was higher in the placenta than in the periphery, suggesting differences in immune cell profiles between the two blood compartments. Furthermore, the percentages of peripheral CD45RO memory-like and CD4 T cells were significantly elevated in aparasitemic women compared to levels in the parasitemic group, with aparasitemic multigravid women having the highest percentages of CD45RO memory-like and CD4 T cells. In contrast, at the placental level, IFN-gamma production by innate NK cells was significantly increased in aparasitemic women compared to parasitemic women, regardless of gravidity. These results suggest that the elevated IFN-gamma-producing NK cells in the placenta and CD45RO memory-like and CD4 T cells in peripheral blood may be involved in protection against malaria infection in pregnancy.     

NK cells exacerbate the pathology of influenza virus infection in mice

NK cells offer a first line of defense against viruses and are considered beneficial to the host during infection. Nevertheless, little is understood regarding the phenotype and function of NK cells in the lung during influenza virus infection. We found that the frequency of NK cells in mouse lung increased during influenza infection, with the majority of a mature phenotype. Cell surface CD107a and intracellular IFN‐γ were detected in cells expressing multiple NK‐cell receptors in infected lung, suggesting that NK cells were activated during infection. The activating receptor NKp46 was predominantly negative on such cells, possibly as a result of encountering influenza HA. Depletion of NK cells in vivo with anti‐asialo GM1 or anti‐NK1.1 reduced mortality from influenza infection and surviving mice recovered their body weight. Pathology induced by NK cells was only observed with high, not medium or low‐dose influenza infection, indicating that the severity of infection influences NK‐cell‐mediated pathology. Furthermore, adoptive transfer of NK cells from influenza‐infected lung, but not uninfected lung, resulted in more rapid weight loss and increased mortality of influenza‐infected mice. Our results indicate that during severe influenza infection of the lung, NK cells have a deleterious impact on the host, promoting mortality.     

Maternal microchimerism in healthy adults in lymphocytes, monocyte/macrophages and NK cells

During pregnancy some maternal cells reach the fetal circulation. Microchimerism (Mc) refers to low levels of genetically disparate cells or DNA. Maternal Mc has recently been found in the peripheral blood of healthy adults. We asked whether healthy women have maternal Mc in T and B lymphocytes, monocyte/macrophages and NK cells and, if so, at what levels. Cellular subsets were isolated after fluorescence activated cell sorting. A panel of HLA-specific real-time quantitative PCR assays was employed targeting maternal-specific HLA sequences. Maternal Mc was expressed as the genome equivalent (gEq) number of microchimeric cells per 100,000 proband cells. Thirty-one healthy adult women probands were studied. Overall 39% (12/31) of probands had maternal Mc in at least one cellular subset. Maternal Mc was found in T lymphocytes in 25% (7/28) and B lymphocytes in 14% (3/21) of probands. Maternal Mc levels ranged from 0.9 to 25.6 and 0.9 to 25.3 gEq/100,000 in T and B lymphocytes, respectively. Monocyte/macrophages had maternal Mc in 16% (4/25) and NK cells in 28% (5/18) of probands with levels from 0.3 to 36 and 1.8 to 3.2 gEq/100,000, respectively. When compared to fetal Mc, as assessed by quantification of male DNA in women with sons, maternal Mc was substantially less prevalent in all cellular subsets; fetal Mc prevalence in T and B lymphocytes, monocyte/macrophages and NK cells was 58, 75, 50 and 62% (P=0.01, P=0.005, P=0.04, P=0.05) respectively. In summary, maternal Mc was identified among lymphoid and myeloid compartments of peripheral blood in healthy adult women. Maternal Mc was less frequent than fetal Mc in all cellular subsets tested. Studies are needed to investigate the immunological effects and function of maternal Mc and to explore whether maternal Mc in cellular subsets has biological effects on her progeny.     

Antibody response to influenza vaccination in healthy adults

The aim of this study was to assess antibody response in 184 healthy adults vaccinated with split influenza vaccine (Begrivac, Chiron Behring). Response to hemagglutinin and neuraminidase was assessed before vaccination and after 1 month by hemagglutination inhibition test and neuraminidase inhibition test. After vaccination, statistically significant increases of antibody titers, both for hemagglutinins and for neuraminidases, were observed. The post-vaccination proportion of persons with protective antihemagglutinin antibody titers ranged from 78.4%to 90.8%, while the proportion of persons with at least a fourfold increase of antihemagglutinin antibody titers ranged from 50.5% to 71.2%. All requirements of the Committee for Proprietary Medicinal Products regarding humoral response to inactivated influenza vaccine in healthy adults were fulfilled. Due to a wide range of age of the persons included in this study, the results were also analyzed in two age groups: from 20 to 45 years and from 46 to 56 years. Nevertheless, there were no statistically significant differences in antibody response between these two groups either for hemagglutinin or for neuraminidase.     

Fc gamma RII-dependent sensitisation of natural interferon-producing cells for viral infection and interferon-alpha responses

Natural interferon-producing cells (NIPC), also called plasmacytoid dendritic cells, are the most potent producers of IFN-alpha in response to viral and bacterial components, serving an important function in innate immune defences. The present work demonstrates that NIPC responsiveness can be primed by immunisation, increasing their capacity to produce IFN-alpha after viral infection. NIPC isolated from pigs immunised against classical swine fever virus (CSFV), a member of the Flaviviridae, were more receptive to viral infection and produced higher levels of IFN-alpha than NIPC from immunologically naive animals. This sensitisation of NIPC was maintained for at least 8 months after immunisation. IFN-alpha production was dependent on live virus and required replication, and the "immune" NIPC responded to lower infectious doses of virus. Co-localisation of the virus with Fc(gamma)RII (CD32) in polarised structures was observed with "immune" NIPC only. This Fc(gamma)RII-dependent virus capture and sensitisation of NIPC, evidently mediated through cytophilic CSFV-specific antibodies, was inhibited by non-specifically aggregated immunoglobulin as well as by pre-formed virus-antibody complexes. In conclusion, these results demonstrate that NIPC not only represent a major player in innate immunity but are also functionally linked to the immunological memory of the adaptive immune system.     

Evaluation of alloreactivity in responder-stimulator pairs by determination of gamma interferon-producing cells and cytotoxic-T-lymphocyte precursor frequencies

We used the enzyme-linked immunospot (ELISPOT) assay and the cytotoxic-T-lymphocyte precursor frequency assay to evaluate alloreactivity in responder-stimulator pairs. High frequencies of responder cells among cells from HLA-mismatched pairs and low frequencies among cells from pairs of siblings with identical HLA types were detected by both assays. The ELISPOT assay thus illustrated the helper and cytotoxic-T-cell response to allogeneic HLA antigens.     

Establishing the reference intervals of NK cell functions in healthy adults

Natural killer (NK) cells play a key role in host defense against microbial pathogens. Establishing the reference intervals (RIs) of NK cell functions would be valuable in assessing the immune status of hosts. We evaluated the NK cell activity in healthy adults. We further established and validated the RIs of representative NK cell functions. Flow cytometry was used to evaluate the cytokine production and CD107a degranulation of NK cells. Levels of soluble IFN-γ in the culture supernatants were evaluated by ELISA. Our results demonstrated that the intracellular IFN-γ production of NK cells was positively correlated with CD107a expression and soluble IFN-γ levels. There were no significant differences in NK cell functions between different age and gender groups. The mean values and RIs of representative NK cell functions are as following: IFN-γ⁺ NK cells (%): 28.09 (11.3–51.95); CD107a⁺ NK cells (%): 17.90 (9.852–27.56); soluble IFN-γ (pg/ml): 330.4 (41.38–717.8). In addition, the intracellular IFN-γ production and degranulation activity of NK cells in patients with colorectal cancer were significantly lower than that in healthy adults. Our study has established the RIs of NK cell functions in healthy adults, which might be used for monitoring the immune status of the hosts.     

Memory T-cell immune response in healthy young adults vaccinated with live attenuated influenza A (H5N2) vaccine

Cellular immune responses of both CD4 and CD8 memory/effector T cells were evaluated in healthy young adults who received two doses of live attenuated influenza A (H5N2) vaccine. The vaccine was developed by reassortment of nonpathogenic avian A/Duck/Potsdam/1402-6/68 (H5N2) and cold-adapted A/Leningrad/134/17/57 (H2N2) viruses. T-cell responses were measured by standard methods of intracellular cytokine staining of gamma interferon (IFN-γ)-producing cells and a novel T-cell recognition of antigen-presenting cells by protein capture (TRAP) assay based on the trogocytosis phenomenon, namely, plasma membrane exchange between interacting immune cells. TRAP enables the detection of activated trogocytosis-positive T cells after virus stimulation. We showed that two doses of live attenuated influenza A (H5N2) vaccine promoted both CD4 and CD8 T-memory-cell responses in peripheral blood of healthy young subjects in the clinical study. Significant differences in geometric mean titers (GMTs) of influenza A (H5N2)-specific IFN-γ(+) cells were observed at day 42 following the second vaccination, while peak levels of trogocytosis(+) T cells were detected earlier, on the 21st day after the second vaccination. The inverse correlation of baseline levels compared to postvaccine fold changes in GMTs of influenza-specific CD4 and CD8 T cells demonstrated that baseline levels of these specific cells could be considered a predictive factor of vaccine immunogenicity.     

Fluctuation of virus antibody levels in healthy adults

In order to determine fluctuations in virus antibody levels, blood specimens were collected weekly from 13 healthy adults during a nine-week period and tested by enzyme immunoassay for IgG antibodies against measles, rubella and cytomegalovirus. In 29 of 35 subject-titer pairs the difference between the highest and lowest titers observed was less than twofold, in five cases it was between twofold and threefold, and in one case it was 3.3-fold. These differences were higher than those caused by intra-assay variation alone. The results indicate that some physiological fluctuations seem to occur in the antibody levels of healthy individuals. Although the fluctuations are usually small, care should be taken if titer increases less than fourfold are used as a criterion to indicate recent infection.     

Contribution of NK, NK T, gamma delta T, and alpha beta T cells to the gamma interferon response required for liver protection against Trypanosoma cruzi

In the present work, we show that intracellular Trypanosoma cruzi is rarely found in the livers of acutely infected mice, but inflammation is commonly observed. The presence of numerous intrahepatic amastigotes in infected gamma interferon (IFN-gamma)-deficient mice corroborates the notion that the liver is protected by an efficient local immunity. The contribution of different cell populations was suggested by data showing that CD4- and CD8-deficient mice were able to restrain liver parasite growth. Therefore, we have characterized the liver-infiltrating lymphocytes and determined the sources of IFN-gamma during acute T. cruzi infection. We observed that natural killer (NK) cells increased by day 7, while T and B cells increased by day 14. Among CD3+ cells, CD4+, CD8+, and CD4- CD8- cell populations were greatly expanded. A large fraction of CD3+ cells were positive for PanNK, a beta1 integrin expressed by NK and NK T cells. However, these lymphocytes were not classic NK T cells because they did not express NK1.1 and showed no preferential usage of Vbeta8. Otherwise, liver NK T (CD3+ NK1.1+) cells were not increased in acutely infected mice. The majority of PanNK+ CD4+ and PanNK+ CD8+ cells expressed T-cell receptor alphabeta (TCRalphabeta), whereas PanNK+ CD4- CD8- cells were positive for TCRgammadelta. In fact, gammadelta T cells showed the most remarkable increase (40- to 100-fold) among liver lymphocytes. Most importantly, intracellular analysis revealed high levels of IFN-gamma production at day 7 by NK cells and at day 14 by CD4+, CD8+, and CD4- CD8- TCRgammadelta+ cells. We concluded that NK cells are a precocious source of IFN-gamma in the livers of acutely infected mice, and, as the disease progresses, conventional CD4+ and CD8+ T cells and gammadelta T cells, but not classic NK-T cells, may provide the IFN-gamma required for liver protection against T. cruzi.     

Therapeutic activity of NK cells against tumors

While it is generally accepted that natural killer (NK) cells, by killing tumor cells in the circulation, represent a first line of defense against metastases, their therapeutic activity against established tumors has been limited. In this review, we describe studies to improve the therapeutic effectiveness of activated NK cells in both animal models and clinical trials to better understand the biological problems that limit their effectiveness.     

Antibody response to influenza vaccine in adults vaccinated with identical vaccine strains in consecutive years

Fifty seven hospital workers received influenza vaccine in November 2003, and the serum HI antibody titer was determined before, 2 and 4 weeks after the vaccination. Thirty seven were vaccinated in November, 2002 consecutively (the repeated vaccination group), and the remaining 20 had not been vaccinated in the previous year (the single vaccination group). Six of the repeated vaccination group received both influenza and hepatitis B vaccination in September, 2004 and the antibody responses were examined 2 weeks later. Two and four weeks after the 2003-vaccination, the HI antibody titers to A/H1N1, A/H3N2, and B in the repeated vaccination group were significantly lower than in the single vaccination group (P < 0.05). This phenomenon had no relation to the pre-vaccination HI antibody titer. The antibody response was low to repeated influenza vaccination, but normal to hepatitis B vaccine in six subjects who had a second vaccination in 2004, showing that this depressed response was influenza-specific. These results suggest that the decreased HI antibody response to repeated influenza vaccination was affected mainly by the previous vaccination per se rather than by the pre-existing antibody titer.     

Drugs active against influenza virus infection in mice

Summary The results obtained with two drugs that were found to be effective against influenza virus infection in mice are reported.The two drugs are C. V. 57455 (4-biphenylylglyoxal bisulfite) and C. V. 58903 (-oxo--(4-biphenylyl)--ethoxy--(4-carboxy-phenyl) amineethane).The drugs were active after administration by the subcutaneous, the oral or the nasal routes prior to and partly also 24 hours after, but not 48 hours after instillation of 5 LD₅₀ of the virus.The drugs were active in low doses providing for an ample margin between the toxic and effective range.The hemagglutinin titers of the lungs of mice which died despite treatment were lower than those found in the lungs of untreated mice.The hemagglutinin titers of the lungs of surviving, unaffected, drugtreated mice were very low or undetectable.The surviving drug-treated mice were resistant to reinfection 2 to 6 weeks later.The results were communicated in part to the Accademia Nazionale dei XL, Rome-Italy.     

Antigen specificity and frequency of autologous and allogeneic helper T cells in the in vitro production of antibody against influenza virus by human blood lymphocytes

The frequency of antigen-specific helper T cells in human peripheral blood mononuclear cells was determined by limiting dilution analysis of specific in vitro antibody responses to influenza virus A/X-31 (A-H3N2). Limiting numbers of irradiated E rosette-forming (E⁺) (T) cells were added to a constant number of syngeneic non-rosette-forming (E^?) (“B”) cells and their ability to support production of antibody to influenza virus determined. The frequency of T helper cells was then calculated by a Poisson distribution analysis and found to range between 0.78 × 10^?5 and 2.86 × 10^?5. Specific antibody production can be obtained in this system from allogeneic combination of E^? and E⁺ cells provided that the added E⁺ cells are irradiated to abrogate alloactivated T suppressor effects. Limiting dilution analysis applied to determine the frequency of helper T cells under these conditions showed that in most, but not all, cases the frequency of T helper cells was higher in allogeneic combinations. This result could be interpreted as showing an increase in the number of activated specific T helper cells, due perhaps to a positive allogeneic effect. On the other hand, it could also be explained by the alloactivation of an entirely different subset of nonspecific helper cells, or by the production of a nonspecific allogeneic helper factor. The specificity of T cell help in allogeneic combinations was therefore examined by adding limiting numbers of E^? cells to a fixed number of E^? cells and simultaneously challenging with two non-cross-reacting influenza viruses A/X-31 and B/HK. Under these conditions, individual cultures produced antibody to A/X-31, B/HK or both. The frequency of cultures producing antibody to both viruses was as predicted for the chance occurrence of specific helper T cells for both antigens being present in the same culture. The fact that a significant number of individual cultures made antibody to only one antigen in both autologous and allogeneic combinations showed that T cell help was antigen-specific in both situations. Thus, for human in vitro antibody responses, antigen-specific T cell help can be obtained across a major histocompatibility (complex) barrier.