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Effects of extrinsic and intrinsic factors on age-associated decline in natural killer cell activity during primary influenza infection
Abstract
The fastest growing segment of the U.S. population is the group of individuals over the age of 65 years. Aging is accompanied by numerous changes in immune function and is associated with an increased morbidity as well as mortality to virus infections. Influenza is a major public health concern. Each year seasonal influenza disease occurs with a number of hospitalizations. In addition, influenza and its secondary complications rank as the 4th leading cause of deaths among the elderly. An age-associated decrease and delay in cell-mediated response, a reduction in the proliferative T cell response, and an altered cytokine production in response to influenza vaccination or infection have been reported. These are parameters of adaptive immune response. However, study of innate immune response against primary influenza infection has lacked attention. Natural killer (NK) cells are an integral part of innate immunity and play a key role in controlling viral infections and foreign pathogens. Here we report that NK cells are essential for controlling influenza virus replication early during infection independent of age. Importantly, there is an age-associated impairment in influenza-inducible NK cytotoxicity and a lack of increase in NK cells at the site of infection. In addition, aged mice produced less interleukin (IL)-15 and interferon (IFN)-γ compared to young mice following influenza infection. While enhancement of NK cytotoxicity in response to stimulation with IL-2 or IL-15 in vitro was comparable in young and aged mice, aged mice demonstrated significantly lower enhancement of granzyme B expression in response to IL-2 or IL-15 and of IFN-γ production in response to IL-15. These results demonstrate that age-associated impairment in influenza-inducible NK cytotoxicity may be caused by a combination of extrinsic influences, i.e. limited NK stimulating cytokines in the aged environment, and intrinsic defects, i.e. a limited inability of NK cells of aged mice to respond to cytokines and possibly other stimuli. These findings suggest that NK cells may be therapeutic targets for an intervention to restore or boost inducible NK cell cytotoxicity during primary viral infection.
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IL-2 and IL-15 are lymphocyte growth factors produced by different cell types with overlapping functions in immune responses. Both cytokines costimulate lymphocyte proliferation and activation, while IL-15 additionally promotes the development and survival of NK cells, NKT cells, and intraepithelial lymphocytes. We have investigated the effects of IL-2 and IL-15 on proliferation, cytotoxicity, and cytokine secretion by human PBMC subpopulations in vitro. Both cytokines selectively induced the proliferation of NK cells and CD56+ T cells, but not CD56− lymphocytes. All NK and CD56+ T cell subpopulations tested (CD4+, CD8+, CD4−CD8−, αβTCR+, γδTCR+, CD16+, CD161+, CD158a+, CD158b+, KIR3DL1+, and CD94+) expanded in response to both cytokines, whereas all CD56− cell subpopulations did not. Therefore, previously reported IL-15-induced γδ and CD8+ T cell expansions reflect proliferations of NK and CD56+ T cells that most frequently express these phenotypes. IL-15 also expanded CD8α+β− and Vα24Vβ11 TCR+ T cells. Both cytokines stimulated cytotoxicity by NK and CD56+ T cells against K562 targets, but not the production of IFN-γ, TNF-α, IL-2, or IL-4. However, they augmented cytokine production in response to phorbol ester stimulation or CD3 cross-linking by inducing the proliferation of NK cells and CD56+ T cells that produce these cytokines at greater frequencies than other T cells. These results indicate that IL-2 and IL-15 act at different stages of the immune response by expanding and partially activating NK receptor-positive lymphocytes, but, on their own, do not influence the Th1/Th2 balance of adaptive immune responses.
C57BL/6 mice genetically deficient in interleukin 15 (IL-15−/− mice) were generated by gene targeting. IL-15−/− mice displayed marked reductions in numbers of thymic and peripheral natural killer (NK) T cells, memory phenotype CD8+ T cells, and distinct subpopulations of intestinal intraepithelial lymphocytes (IELs). The reduction but not absence of these
populations in IL-15−/− mice likely reflects an important role for IL-15 for expansion and/or survival of these cells. IL-15−/− mice lacked NK cells, as assessed by both immunophenotyping and functional criteria, indicating an obligate role for IL-15
in the development and functional maturation of NK cells. Specific defects associated with IL-15 deficiency were reversed
by in vivo administration of exogenous IL-15. Despite their immunological defects, IL-15−/− mice remained healthy when maintained under specific pathogen-free conditions. However, IL-15−/− mice are likely to have compromised host defense responses to various pathogens, as they were unable to mount a protective
response to challenge with vaccinia virus. These data reveal critical roles for IL-15 in the development of specific lymphoid
lineages. Moreover, the ability to rescue lymphoid defects in IL-15−/− mice by IL-15 administration represents a powerful means by which to further elucidate the biological roles of this cytokine.
Recent evidence suggests that NK cells require priming to display full effector activity. In this study, we demonstrate that IL-18 contributed to this phenomenon. IL-18 signaling-deficient NK cells were found to be unable to secrete IFN-gamma in response to ex vivo stimulation with IL-12. This was not due to a costimulatory role of IL-18, because blocking IL-18 signaling during the ex vivo stimulation with IL-12 did not alter IFN-gamma production by wild-type NK cells. Rather, we demonstrate that IL-18 primes NK cells in vivo to produce IFN-gamma upon subsequent stimulation with IL-12. Importantly, IL-12-induced IFN-gamma transcription by NK cells was comparable in IL-18 signaling-deficient and -sufficient NK cells. This suggests that priming by IL-18 leads to an improved translation of IFN-gamma mRNA. These results reveal a novel type of cooperation between IL-12 and IL-18 that requires the sequential action of these cytokines.
Natural killer (NK) cells are important effector cells in the control of infections. The cellular and molecular signals required for NK cell activation in vivo remain poorly defined. By using a mouse model for the inducible ablation of dendritic cells (DCs), we showed that the in vivo priming of NK cell responses to viral and bacterial pathogens required the presence of CD11c(high) DCs. After peripheral Toll-like receptor (TLR) stimulation, NK cells were recruited to local lymph nodes, and their interaction with DCs resulted in the emergence of effector NK cells in the periphery. NK cell priming was dependent on the recognition of type I IFN signals by DCs and the subsequent production and trans-presentation of IL-15 by DCs to resting NK cells. CD11c(high) DC-derived IL-15 was necessary and sufficient for the priming of NK cells. Our data define a unique in vivo role of DCs for the priming of NK cells, revealing a striking and previously unappreciated homology to T lymphocytes of the adaptive immune system.
The 2008 influenza season was moderate overall, with fewer laboratory-confirmed cases and influenza-like illness (ILI) presentations than in 2007, which was the most severe influenza season since national reporting of influenza began in 2001. In 2008, the number of laboratory-confirmed notifications for influenza was 1.9 times higher than the 5-year mean. High notification rates were reflected in an increase in presentations with ILI to sentinel general practices and emergency departments. Notification rates were highest in the 0-4 year age group. Unusually, the season was predominantly due to influenza B, with 54% of notifications being influenza B and 43% being influenza A (3% type unknown). The rate of influenza B was higher among the younger age groups, compared with influenza A, which was more common in the older age groups. Of influenza viruses circulating during the 2008 season, A(H3) viruses were predominant and were antigenically similar to the 2008 A(H3) vaccine strain, while the majority of A(H1) strains showed significant drift away from the 2008 A(H1) vaccine strain. There were approximately equal proportions of viruses from the 2 influenza B lineages B/Yamagata and B/Victoria.
Each year, influenza virus infection causes severe morbidity and mortality, particularly in the most susceptible groups including children, the elderly (>65 years-old) and people with chronic respiratory diseases. Among the several factors that contribute to the increased susceptibility in elderly populations are the higher prevalence of chronic diseases (e.g. diabetes) and the senescence of the immune system.
In this study, aged and adult mice were infected with sublethal doses of influenza virus (A/Puerto Rico/8/1934). Differences in weight loss, morbidity, virus titer and the kinetics of lung infiltration with cells of the innate and adaptive immune responses were analyzed. Additionally, the main cytokines and chemokines produced by these cells were also assayed.
Compared to adult mice, aged mice had higher morbidity, lost weight more rapidly, and recovered more slowly from infection. There was a delay in the accumulation of granulocytic cells and conventional dendritic cells (cDCs), but not macrophages in the lungs of aged mice compared to adult animals. The delayed infiltration kinetics of APCs in aged animals correlated with alteration in their activation (CD40 expression), which also correlated with a delayed detection of cytokines and chemokines in lung homogenates. This was associated with retarded lung infiltration by natural killer (NK), CD4+ and CD8+ T-cells. Furthermore, the percentage of activated (CD69+) influenza-specific and IL-2 producer CD8+ T-cells was higher in adult mice compared to aged ones. Additionally, activation (CD69+) of adult B-cells was earlier and correlated with a quicker development of neutralizing antibodies in adult animals.
Overall, alterations in APC priming and activation lead to delayed production of cytokines and chemokines in the lungs that ultimately affected the infiltration of immune cells following influenza infection. This resulted in delayed activation of the adaptive immune response and subsequent delay in clearance of virus and prolonged illness in aged animals. Since the elderly are the fastest growing segment of the population in developed countries, a better understanding of the changes that occur in the immune system during the aging process is a priority for the development of new vaccines and adjuvants to improve the immune responses in this population.
Expression of inhibitory killer cell immunoglobulin-like receptors (KIRs) specific for self-major histocompatibility complex (MHC) class I molecules provides an educational signal that generates functional natural killer (NK) cells. However, the effects of activating KIRs specific for self-MHC class I on NK-cell education remain elusive. Here, we provide evidence that the activating receptor KIR2DS1 tunes down the responsiveness of freshly isolated human NK cells to target cell stimulation in donors homozygous for human leukocyte antigen (HLA)-C2, the ligand of KIR2DS1. The tuning was apparent in KIR2DS1(+) NK cells lacking expression of inhibitory KIRs and CD94/NKG2A, as well as in KIR2DS1(+) NK cells coexpressing the inhibitory MHC class I-specific receptors CD94/NKG2A and KIR2DL3, but not KIR2DL1. However, the tuning of responsiveness was restricted to target cell recognition because KIR2DS1(+) NK cells responded well to stimulation with exogenous cytokines. Our results provide the first example of human NK-cell education by an activating KIR and suggest that the education of NK cells via activating KIRs is a mechanism to secure tolerance that complements education via inhibitory KIRs.
The genetic make-up of the host has a major influence on its response to combat pathogens. For influenza A virus, several single gene mutations have been described which contribute to survival, the immune response and clearance of the pathogen by the host organism. Here, we have studied the influence of the genetic background to influenza A H1N1 (PR8) and H7N7 (SC35M) viruses. The seven inbred laboratory strains of mice analyzed exhibited different weight loss kinetics and survival rates after infection with PR8. Two strains in particular, DBA/2J and A/J, showed very high susceptibility to viral infections compared to all other strains. The LD(50) to the influenza virus PR8 in DBA/2J mice was more than 1000-fold lower than in C57BL/6J mice. High susceptibility in DBA/2J mice was also observed after infection with influenza strain SC35M. In addition, infected DBA/2J mice showed a higher viral load in their lungs, elevated expression of cytokines and chemokines, and a more severe and extended lung pathology compared to infected C57BL/6J mice. These findings indicate a major contribution of the genetic background of the host to influenza A virus infections. The overall response in highly susceptible DBA/2J mice resembled the pathology described for infections with the highly virulent influenza H1N1-1918 and newly emerged H5N1 viruses.
The mammalian immune response to infection is mediated by 2 broad arms, the innate and adaptive immune systems. Innate immune cells are a first-line defense against pathogens and are thought to respond consistently to infection, regardless of previous exposure, i.e., they do not exhibit memory of prior activation. By contrast, adaptive immune cells display immunologic memory that has 2 basic characteristics, antigen specificity and an amplified response upon subsequent exposure. Whereas adaptive immune cells have rearranged receptor genes to recognize the universe of antigens, natural killer (NK) cells are innate immune lymphocytes with a limited repertoire of germ-line encoded receptors for target recognition. NK cells also produce cytokines such as IFN-gamma (IFN-gamma) to protect the host during the innate response to infection. Herein, we show that cytokine-activated NK cells transferred into naïve hosts can be specifically detected 7-22 days later when they are phenotypically similar to naïve cells and are not constitutively producing IFN-gamma. However, they produce significantly more IFN-gamma when restimulated. This memory-like property is intrinsic to the NK cell. By contrast, memory-like NK cells do not express granzyme B protein and kill targets similarly to naïve NK cells. Thus, these experiments identify an ability of innate immune cells to retain an intrinsic memory of prior activation, a function until now attributed only to antigen-specific adaptive immune cells.
Energy restriction (ER) without malnutrition extends lifespan in mice and postpones age-related changes in immunity. However, we have previously shown that aged (22 mo old) ER mice exhibit increased mortality, impaired viral clearance, and reduced natural killer (NK) cell cytotoxicity following influenza infection compared with aged mice that consumed food ad libitum (AL). To determine whether the detrimental effects of ER in response to influenza infection occur independently of advanced age, young adult (6 mo) male C57BL/6 mice consuming an AL or ER diet were infected with influenza A virus (H1N1, PR8). Young adult ER mice exhibited increased mortality (P < 0.05) and weight loss (P < 0.01) in response to infection. ER mice exhibited decreased total (P < 0.001) and NK1.1+ lymphocytes (P < 0.05) in lung and reduced influenza-induced NK cell cytotoxicity in both lung (P < 0.01) and spleen (P < 0.05). Importantly, the mRNA expression of interferon (IFN)alpha/beta (P < 0.05) was also reduced in the lungs of ER mice in response to infection, and in vitro stimulation of NK cells from ER mice with type I IFN resulted in cytotoxicity comparable to that in NK cells from AL mice. In contrast, NK cell activation was enhanced in ER mice, determined as an increase in the percentage of NK cells expressing B220 (P < 0.001) and increased intracellular production of IFNgamma (P < 0.01). These data describe an age-independent and detrimental effect of ER on the innate immune response to influenza infection and suggest that a decrease in NK cell number and alterations in the NK cell-activating environment may contribute to decreased innate immunity in ER mice.
We have studied the cell-surface phenotype of natural killer (NK) cells of NZB and B6 mice which react to an MuLV+ lymphoid tumor. (a) NK cells do not express Thy1, Ly2, or Ig surface markers. (b) NK cells express an antigen recognized by C3H anti-CE antiserum ('anti-Ly1.2 antiserum'). Inasmuch as NK activity of spleen cells from B6 and B6/Ly1.1 congenic strains were both equally sensitive to C3H anti-CE antiserum, the NK antigen is distinct from Ly1.2. This point was confirmed by the observation that alphaNK activity was removed by absorption of C3H anti-CE antiserum with spleen cells from either B6 or B6/Ly1.1 congenic strains. Absorption of C3H alphaCE serum with BALB/c thymocytes and spleen cells (which are Ly1.2+NK-) removed anti-Ly1.2 activity and left anti-NK activity intact. This absorption step could be circumvented by inserting the BALB/c genotype into the recipient immunized to CE cells (i.e., (C3H X BALB/c)F1 alphaCE spleen cells). This antiserum, provisionally termed 'anti-NK', defines a new subclass of lymphocytes which may play a central role in the immunosurveillance against tumors.
Spleen cells from 2- to 3-month-old normal mice of some strains having a low incidence of spontaneous leukemia were found to lyse cells of the spontaneous AKR leukemia K36 in the 51Cr release assay. Incubation of 51Cr-labeled ADR K36 cells with spleen cells from normal C57BL/6, C57L, C57BL/10, and RF mice resulted in the release of significantly more 51Cr than that released in the presence of medium alone. In contrast, 51Cr released from AKR K36 cells after incubation with spleen cells from mice of the high leukemic strains AKR and C58 was less than that released spontaneously. The results of competitive inhibition tests when C57BL/6 spleen cells were incubated simultaneously with 51Cr-labeled AKR K36 target cells and varying numbers of nonlabeled cells demonstrated that the cytotoxic activity of normal C57BL/6 spleen cells was directed against an antigen(s) associated with several leukemias, but that was undetectable on normal thymocytes. Pretreatment of C57BL/6 spleen cells with carbonyl iron and a magnet, which removed phagocytic macrophages, did not decrease the cytotoxic acitivity for AKR K36 cells.
In the present report we describe a CD4+8- heat stable antigen-negative (HSA-) thymocyte subpopulation that expresses a distinguishably low density of alpha beta T-cell antigen receptors (TCRlo) from the majority of CD4+8- high-density TCR (TCRhi) mature-type thymocytes. This subpopulation appears relatively late in life. Analysis of MEL-14, Pgp-1 (CD44), ICAM-1 (CD54), and NK1.1 expression on this subpopulation revealed that the CD4+8- TCRlo population was a population having unique characteristics (MEL-14-, CD44+, ICAM-1+, and NK1.1+) compared to the CD4+8- TCRhi thymocytes, most of which are MEL-14+, CD44-, ICAM-1-, and NK1.1-. When TCR beta-chain variable region (V beta) usage was analyzed, this thymic population expressed predominantly products of V beta 7 and V beta 8.2 TCR gene families. Interestingly, cells with V beta 8.1 TCRs, which are reactive to Mls-1a antigens, were not eliminated from the CD4+8- HSA- TCRlo subpopulation but had been eliminated from the major CD4+8- HSA- TCRhi subpopulation in Mls-1a strains. A subset with a phenotype similar to the CD4+8- HSA- TCRlo thymocytes was also identified primarily in bone marrow, and this subset constituted approximately half of the CD4+ T cells in the bone marrow. The CD4+8- HSA- TCRlo cells showed extremely high proliferative responses to immobilized anti-TCR antibody but generated negligible responses to allogeneic H-2 antigens compared to the responses generated by the major CD4+8- HSA- CD3hi cells. However, the CD4+8- HSA- TCRlo cells in Mls-1b mice mounted vigorous proliferative responses to Mls-1a antigens but not in Mls-1a mice. The properties of this T-cell subset suggest that these cells belong to a lineage distinct from the major T-cell population.
During the innate immune response to infection, monocyte-derived cytokines (monokines), stimulate natural killer (NK) cells to produce immunoregulatory cytokines that are important to the host's early defense. Human NK cell subsets can be distinguished by CD56 surface density expression (ie, CD56bright and CD56dim). In this report, it is shown that CD56bright NK cells produce significantly greater levels of interferon-γ, tumor necrosis factor-β, granulocyte macrophage–colony-stimulating factor, IL-10, and IL-13 protein in response to monokine stimulation than do CD56dim NK cells, which produce negligible amounts of these cytokines. Further, qualitative differences in CD56bright NK-derived cytokines are shown to be dependent on the specific monokines present. For example, the monokine IL-15 appears to be required for type 2 cytokine production by CD56bright NK cells. It is proposed that human CD56bright NK cells have a unique functional role in the innate immune response as the primary source of NK cell–derived immunoregulatory cytokines, regulated in part by differential monokine production.
The murine Ly-49 antigen belongs to a family of type II transmembrane molecules containing lectin-like domains. The original member of this family, Ly-49A, has been demonstrated to be expressed by a subpopulation of natural killer (NK) cells, bind certain class I major histocompatibility complexes (MHC), and act as a negative regulator of lytic activity. The expression patterns and functional activities of the other Ly-49s, however, is unknown. We extended the study of this family by isolating cDNAs encoding two new Ly-49 molecules. The reactivity of these and previously identified Ly-49 molecules with NK antibodies was tested in a COS cell expression system. YE1/32 and YE1/48 bound Ly-49A specifically, and 5E6 reacted only with Ly-49C. Three-color flow cytometric analysis demonstrated Ly-49A and Ly-49C expression defines complex, but distinct subsets within NK1.1+ cells. Some NK1.1-CD3+ as well as NK1.1-CD3- cells expressing Ly-49A or C were also detected. Analysis of MHC congenic strains of mice demonstrated that YE1/32+ and YE1/48+ NK cells are not deleted, as has been shown with the Ly-49A mAb A1. Furthermore, COS cells transfected with Ly-49A bound H-2d and H-2k cell lines, whereas Ly-49C transfectants bound H-2d, H-2k, H-2b, and H-2s. The antibodies 5E6 and 34-1-2S (anti-class I MHC) inhibited the binding of Ly-49C to an H-2s cell line. These results imply that the NK cell antigens Ly-49A and C bind to different repertoires of class I MHC molecules.
The regulation of human natural killer (NK) cell activation is under the control of a network of regulatory signals provided by cytokines. In the present study, we investigated the functional interaction between interleukin (IL)-4 and two monocyte/macrophage-derived cytokines, IL-12 and IL-15, during the process of NK stimulation. Using freshly isolated human NK cells, we have demonstrated that IL-4 negatively regulates lymphokine-activated killer (LAK) activity induced by IL-15 against the NK-resistant Daudi target cells. In contrast, IL-4 had no effect on IL-12-stimulated LAK generation. The differential effect of IL-4 on NK cell activation by IL-12 and IL-15 correlates with its ability to increase or to down-regulate the level of tumor necrosis factor-alpha and interferon-gamma release by NK cells, respectively. In contrast, endogenous transforming growth factor-beta 1 does not appear to be involved in the IL-4 regulatory pathway. Furthermore, while IL-4 was found to decrease the basal expression of the IL-2 receptor beta subunit utilized by IL-15, it had no effect on the expression of the beta 1 chain of the IL-12 receptor compared to untreated cells. Northern blot analysis indicated that the IL-4 regulatory effect on NK lytic function was associated with its capacity to down-regulate granzyme B and perforin gene transcription in response to IL-15 and its failure to affect the expression of both gene's in response to IL-12. Together, these data suggest the existence of a distinct cross-talk between IL-4 and IL-15 or IL-12 signaling pathways during the regulation of human non-major histocompatibility complex-restricted cytotoxicity.
Subsets of T and natural killer (NK) lymphocytes express the CD94-NKG2A heterodimer, a receptor for major histocompatibility complex class I molecules. We show here that engagement of the CD94-NKG2A heterodimer inhibits both antigen-driven tumor necrosis factor (TNF) release and cytotoxicity on melanoma-specific human T cell clones. Similarly, CD16-mediated NK cell cytotoxicity is extinguished by cross-linking of the CD94-NKG2A heterodimer. Combining in vivo and in vitro analysis, we report that both I/VxYxxL immunoreceptor tyrosine-based inhibition motifs (ITIM) present in the NKG2A intracytoplasmic domain associate upon tyrosine phosphorylation with the protein tyrosine phosphatases SHP-1 and SHP-2, but not with the polyinositol phosphatase SHIP. Determination of the dissociation constant, using surface plasmon resonance analysis, indicates that NKG2A phospho-ITIM interact directly with the SH2 domains of SHP-1 and SHP-2 with a high affinity. Engagement of the CD94-NKG2A heterodimer therefore appears as a protein-tyrosine phosphatase-based strategy that negatively regulates both antigen-induced T cell response and antibody-induced NK cell cytotoxicity. Our results suggest that this inhibitory pathway sets the threshold of T and NK cell activation.
The Ly-49 family consists of at least nine members, of which Ly-49A and C have been found to be NK-cell inhibitory receptors specific for class I MHC. The functions of other Ly-49 molecules are still unclear. Further analysis of Ly-49 is complicated by the cross-reactivities of some anti-Ly-49 anybodies initially thought to be specific for individual Ly-49 molecules. Studies on the role of Ly-49 in hybrid resistance as well as on allelic exclusion are also complicated by our recent finding that a novel Ly-49CB6 gene is the likely allelic form of Ly-49CMLB AS opposed to a previously reported highly related but distinct gene in B6 mice. In cell binding assays, only Ly-49A and C show significant binding to class I MHC, Ly-49A and C also bind some polysaccharides, and carbohydrates on class I MHC seem to be important for its binding to Ly-49, However, this interaction involves NOT only the carbohydrate recognition domain of Ly-49 but also a part of the stalk region, suggesting that both caxbohydrates and peptide backbone of class I MHC may be recognized by Ly-49. It is likely that additional Ly-49 mole cells yet to be identified function as NK-inhibitory receptors specific for class I MHC.
Influenza virus is a major human pathogen that causes epidemics and pandemics with increased morbidity and, especially in the elderly and those with pre-existing medical conditions, increased mortality. Influenza is characterised by respiratory symptoms and constitutional symptoms. Whilst knowledge of the mechanisms underlying host and tissue specificity has advanced considerably of late we still know relatively little about other aspects of influenza virus virulence. In this review, we will explore what is known about the role of apoptosis in respiratory epithelial cell damage and the role of cytokines in inflammation and constitutional symptoms with particular emphasis on the link between apoptosis, inflammation, fever and cytokine production.
Influenza and respiratory syncytial virus are the two most important causes of viral lower respiratory tract disease. The innate immune response to these viruses, determined by multiple cell types, creates a cytokine milieu that affects the adaptive immune response and disease manifestations. This review addresses the function of cytokines released from cells resident in the respiratory tract after infection with influenza and respiratory syncytial virus, and how differences in early cytokine release may impact disease pathogenesis and host response.
Natural killer (NK) cells are cytotoxic cells that play a critical role in the innate immune response against infections and tumors. Recent studies on NK cell biology have demonstrated that besides their cytotoxic function, NK cells express cytokine and chemokine receptors and also that they secrete other immunoregulatory cytokines and chemokines, supporting their relevance in the regulation of the immune response by promoting downstream adaptive, Th1 mediated, responses against infections. Immunosenescence is the deterioration of the immune response associated with aging. It is characterized mainly by a defective T cell response, but includes changes in the number and function of other cells of the innate immune system. Age-associated alterations in the number and function of NK cells have been reported. There is a general consensus that a progressive increase in the percentage of NK cells with a mature phenotype occurs in elderly donors associated with an impairment of their cytotoxic capacity when considered on a “per cell” basis. The response of NK cells from elderly individuals to IL-2 or other cytokines is also decreased in terms of proliferation, expression of CD69 and killing of NK-resistant cell lines. Furthermore early IFN-γ and chemokine production in response to IL-2 or IL-12 is also decreased. However aging does not significantly alter other NK cell functions such as TNF-α production or perforin induction in response to IL-2. The percentage of T cells that co-express NK cell markers is also increased in aging. These results indicate that the increase in the number of “classical” mature NK and NK/T cells in aging is associated with a defective functional capacity of NK cells. Low NK cell number or function in elderly individuals is associated with increased mortality risk and increased incidence of severe infections, supporting the role of NK cells in the defense against infections in the elderly.
IL-18 is a cytokine that is secreted from activated macrophages and induces IFNgamma production. To investigate the in vivo role of IL-18, we generated IL-18-deficient mice. In Propionibacterium acnes (P. acnes)-primed IL-18-deficient mice, LPS-induced IFNgamma production was markedly reduced, despite normal IL-12 induction. Natural killer cell activity was significantly impaired. Th1 cell response after injection of P. acnes or Mycobacterium bovis (bacillus Calmette-Guerin [BCG]) was significantly reduced. Similar results were observed in IL-12-deficient mice. Interestingly, Th1 response was induced after BCG infection in IL-12-deficient mice. We therefore generated mice lacking both IL-18 and IL-12. In these mice, NK activity and Th1 response were further impaired. This demonstrates the important role of both IL-18 and IL-12 in NK activity, as well as in in vivo Th1 response.
The mechanism by which interferon (IFN) pretreatment of effector cells augments natural killer (NK) cell-mediated cytotoxicity (CMC) was examined by determining whether IFN has any effect on the production of natural killer cytotoxic factors (NKCF). NKCF are released into the supernatant of co-cultures of murine spleen cells and YAC-1 stimulator cells, and their lytic activity is measured against YAC-1 target cells. It was demonstrated that pretreatment of effector cells with murine fibroblast IFN or polyinosinic-polycytidylic acid (pIC) resulted in the release of NKCF with augmented lytic activity. Evidence indicated that the IFN-induced augmentation of NKCF activity required protein synthesis during the IFN pretreatment period, because concurrent pretreatment with both IFN and cycloheximide abrogated the IFN effect. Protein synthesis, however, is not required for the production of base levels of NKCF because emetine pretreatment of normal spleen cells did not result in a decrease in NKCF production. Furthermore, substantial levels of NKCF activity could be detected in freeze-thaw lysates of freshly isolated spleen cells. Cell populations enriched for NK effector cells, such as nylon wool-nonadherent nude mouse spleen cells, produced lysates with high levels of NKCF activity, whereas lysates of CBA thymocytes were devoid of NKCF activity. Pretreatment of spleen cells with either IFN or pIC resulted in an augmentation of the NKCF activity present in their cell lysates. Taken altogether, these findings suggest that freshly isolated NK cells contain preformed pools of NKCF. Pretreatment of these cells with IFN causes de novo synthesis of additional NKCF and/or activation of preexisting NKCF. According to our model for the mechanism of NK CMC, target cell lysis is ultimately the result of transfer of NKCF from the effector cell to the target cell. The evidence presented here suggests that the IFN-induced augmentation of NK activity could be accounted for by an increase in the synthesis, activation, and/or release of NKCF.
Ageing is associated with several alterations in the immune system. Our aim in this study was to compare the development of immunity to Schistosoma mansoni infection in young versus aged C57Bl/6 mice using the liver as the main organ to evaluate pathological alterations and immune responses. In the acute phase, young mice had large liver granulomas with fibrosis and inflammatory cells. Chronic phase in young animals was associated with immunomodulation of granulomas that became reduced in size and cellular infiltrate. On the other hand, aged animals presented granulomas of smaller sizes already in the acute phase. Chronic infection in these mice was followed by no alteration in any of the inflammatory parameters in the liver. In concert with this finding, there was an increase in activated CD4+ T, CD19+ B and NK liver cells in young mice after infection whereas old mice had already higher frequencies of activated B, NK and CD4+ T liver cells and infection does not change these frequencies. After infection, liver production of inflammatory and regulatory cytokines such as IFN-gamma, IL-4 and IL-10 increased in young but not in old mice that had high levels of IL-4 and IL-10 regardless of their infection status. Our data suggest that the unspecific activation status of the immune system in aged mice impairs inflammatory as well as regulatory immune responses to S. mansoni infection in the liver, where major pathological alterations and immunity are at stage. This poor immune reactivity may have a beneficial impact on disease development.
IL-12 is a potent proinflammatory cytokine. The effects of IL-12 are thought to be mediated by IFN-gamma production by NK, NKT, and T cells. In this study, we show that although IL-12 stimulates NK and NK1.1(+) T cells in bulk mouse splenocytes, it does not significantly stimulate purified NK cells, indicating that other cells are required. IL-12 stimulates T cell-deficient spleen cells and those depleted of macrophages. Unexpectedly, the depletion of dendritic cells also has little effect on the stimulation of spleen cells with IL-12. In contrast, B cell depletion almost completely inhibits IL-12-induced IFN-gamma production and B cell-deficient spleen cells are poorly stimulated with IL-12. Furthermore, purified NK cells are stimulated with IL-12 in the presence of purified B cells. Thus, B cells are necessary and also sufficient for the stimulation of purified NK cells with IL-12. Whereas spleen cells from IL-18-deficient mice are not stimulated with IL-12, NK cells purified from IL-18-deficient mice are stimulated with IL-12 in the presence of wild-type (WT) B cells, and WT NK cells are not stimulated with IL-12 in the presence of IL-18-deficient B cells. Cell contact between B and NK cells is also required for IL-12-induced IFN-gamma production. Finally, B cell-deficient mice injected with IL-12 produce significantly less IFN-gamma and IL-18 in the sera than WT mice do. Thus, stimulation of NK cells with IL-12 requires B cell cooperation in vitro as well as in vivo.
The human liver is enriched in NK cells which are potent effectors of the innate immune system. We have determined that liver NK cells freshly isolated from surgical specimens from patients with hepatic malignancy have less cytolytic activity than autologous blood NK cells. This difference was due to a higher proportion of CD16(-) NK cells in the liver and reduced cytotoxicity by CD16(+) liver NK cells compared with their blood counterparts. CD16(+) liver NK cells had similar expression of activating NK receptors and had similar intracellular granzyme B and perforin content compared with CD16(+) blood NK cells. CD16(+) liver NK cells contained a reduced fraction of cells with inhibitory killer Ig-like receptors specific for self-MHC class I (self-killer Ig-related receptor (KIR)) and an increased fraction of self-KIR(neg)NKG2A(pos) and self-KIR(neg)NKG2A(neg) cells. Using single-cell analysis of intracellular IFN-gamma production and cytotoxicity assays, we determined that CD16(+) liver NK cells expressing self-KIR were more responsive to target cells than those cells that did not express self-KIR molecules. CD16(+) liver NK cells gained cytolytic function when stimulated with IL-2 or cultured with LPS or poly(I:C)-activated autologous liver Kupffer cells. Thus, the human liver contains NK cell subsets which have reduced effector function, but under appropriate inflammatory conditions become potent killers.
The immune response to influenza A virus is characterized by an influx of both macrophages and T lymphocytes into the lungs of the infected host, accompanied by induced expression of a number of CC chemokines. CC chemokine receptors CCR5 and CCR2 are both expressed on activated macrophages and T cells. We examined how the absence of these chemokine receptors would affect pulmonary chemokine expression and induced leukocyte recruitment by infecting CCR5-deficient mice and CCR2-deficient mice with a mouse-adapted strain of influenza A virus. CCR5−/− mice displayed increased mortality rates associated with acute, severe pneumonitis, whereas CCR2−/− mice were protected from the early pathological manifestations of influenza because of defective macrophage recruitment. This delay in macrophage accumulation in CCR2−/− mice caused a subsequent delay in T cell migration, which correlated with high pulmonary viral titers at early time points. Infected CCR5−/− mice and CCR2−/− mice both exhibited increased expression of the gene for MCP-1, the major ligand for CCR2−/− and a key regulator of induced macrophage migration. These studies illustrate the very different roles that CCR5 and CCR2 play in the macrophage response to influenza infection and demonstrate how defects in macrophage recruitment affect the normal development of the cell-mediated immune response.
Immunotherapeutic strategies may promote T and/or natural killer (NK) cell cytotoxicity. NK cells have the potential to exert a powerful anti-leukaemia effect, as demonstrated by studies of allogeneic transplantation. We have previously shown that CD80/interleukin 2 (IL2) lentivirus (LV)-transduced AML cells stimulate in-vitro T cell activation. The present study demonstrated that allogeneic and autologous culture of peripheral blood mononuclear cells with CD80/IL2-expressing AML cells also promoted NK cell cytotoxicity. Expression of the activation receptors NKp30, NKp44, CD244, CD25, CD69 and HLA-DR significantly increased following allogeneic culture and a consistent increased expression of NKp30, NKp44, NKp46, NKG2D, NKG2C and CD69, and up-regulation of the cytolytic marker CD107a was detected following autologous culture with LV-CD80/IL2 AML cells. Furthermore, increased NK cell lysis of K562 and primary AML blasts was detected. The lytic activity increased by twofold against K562 (from 46.6% to 90.4%) and allogeneic AML cells (from 11.8% to 20.1%) following in-vitro stimulation by CD80/IL2-expressing AML cells. More importantly for potential therapeutic applications, lysis of primary AML cells by autologous NK cells increased by more than 40-fold (from 0.4% to 22.5%). These studies demonstrated that vaccination of patients with CD80/IL2-transduced AML cells could provide a powerful strategy for T/NK cell-mediated stimulation of anti-leukaemic immunological responses.
Interleukin-15 (IL-15) is a cytokine important for the development, maturation, and function of many cells of the immune system including NK, NKT, gammadeltaT, and CD8(+) T cells. The relationship between IL-15 and B lymphocytes however, is not well characterized and is the focus of our study. Previous in vitro reports have shown that IL-15 increases proliferation of B lymphocytes and increases antibody secretion however, this relationship remains inadequately defined in vivo. The focus of this study was to examine the role of IL-15 in B cell homeostasis and function in vivo using mice that either over express IL-15 (IL-15tg mice) or are deficient in IL-15 (IL-15(-/-) mice) production. Here we report significant differences between the B cell populations of IL-15(-/-), C57BL/6, and IL-15tg mice. In fact, increased expression of IL-15 resulted in a significant decrease in the percentage and absolute number of CD19(+) cells. In vitro B cell co-cultures implicate interferon-gamma (IFN-gamma) as the factor responsible for inhibiting B cell proliferation. We also show that IL-15 expression affects B cell function, as B cells from IL-15 transgenic mice produce greater amounts of IgG and IgA than IL-15 knockout mice in vitro. Interestingly, despite significant differences in B cell numbers in these strains, there were no significant differences in total antibody titers in serum and vaginal washes of these mice. Results from our in vivo and in vitro experiments suggest that altered expression of IL-15 affects B cell homeostasis through the induction of NK cell-derived IFN-gamma.
Supplementation with mushroom-derived active hexose correlated compound (AHCC) modulates immunity and increases survival in response to a broad spectrum of acute infections, including influenza virus infection. However, dose-response data are nonexistent. Therefore, the aims of this study were to evaluate AHCC supplementation at various doses and determine the effects of low-dose supplementation on the immune response in a mouse model of influenza virus infection. We hypothesized that AHCC supplementation would influence the immune response to influenza infection in a dose-dependent manner. Male C57BL/6 mice were supplemented with AHCC at daily doses of 0.05, 0.1, 0.5, and 1 g/kg and infected intranasally with influenza A virus (H1N1, PR8). Supplemented mice demonstrated a dose-dependent increase in survival and reduction in the loss of body weight. To further evaluate the effects of low-dose AHCC supplementation on the immune response to influenza infection, mice were supplemented with 0.1 g/kg per day and infected with a sublethal dose of influenza virus. Supplemented mice exhibited enhanced virus clearance and decreased weight loss compared to controls. Low-dose supplementation did not influence total natural killer (NK) cell cytotoxicity, although lytic efficiency was increased in the spleens of AHCC-supplemented mice, indicating enhanced NK cell function per cell. In conclusion, these data suggest that the effects of AHCC on the immune response to influenza infection are dose dependent and that low-dose AHCC supplementation improves the response to influenza infection despite no effect on total NK cell cytotoxicity.
The impact of influenza has been recognized for centuries. Its seasonality in temperate climates has allowed estimates of mortality and severe morbidity, such as hospitalization, to be made statistically, without identifying cases virologically. Most influenza related mortality occurs in older individuals and those with underlying conditions. In addition to those groups, influenza hospitalizations occur in younger children and pregnant women. Morbidity is more difficult to identify and laboratory confirmation is required for precise estimates to be made. Younger individuals experience the highest frequency of illnesses caused by all subtypes. This has resulted in suggested strategies for community control by vaccinating children.
The inhibitory CD94/NKG2A and activating CD94/NKG2C killer lectin-like receptors specific for HLA-E have been reported to be selectively expressed by discrete NK and T cell subsets. In the present study, minor proportions of NK and T cells coexpressing both CD94/NKG2A and CD94/NKG2C were found in fresh peripheral blood from adult blood donors. Moreover, CD94/NKG2A surface expression was transiently detected upon in vitro stimulation of CD94/NKG2C+ NK cells in the presence of irradiated allogeneic PBMC or rIL-12. A similar effect was observed upon coculture of NKG2C+ NK clones with human CMV-infected autologous dendritic cell cultures, and it was prevented by an anti-IL-12 mAb. NKG2A inhibited the cytolytic activity of NKG2C+ NK clones upon engagement either by a specific mAb or upon interaction with a transfectant of the HLA class I-deficient 721.221 cell line expressing HLA-E. These data indicate that beyond its constitutive expression by an NK cell subset, NKG2A may be also transiently displayed by CD94/NKG2C+ NK cells under the influence of IL-12, providing a potential negative regulatory feedback mechanism.
The development of a cell-mediated immune response to Sendai virus infection in mice was examined by the use of a 51Cr release assay of cytotoxicity. A low level of "background cytotoxicity" to Sendai virus-infected L cells was found in the spleens of uninfected CBA mice. Spleen cells from Sendai-infected mice showed an elevated level of cytotoxicity against these target cells for a period of 5 weeks, commencing 4 days after infection of the mice. A more transient response was observed in the spleens of mice infected with a serologically distinct virus, the Kunz strain of influenza. This cross-reacting, cell-mediated immune response was intermediate between that observed in unsensitized and Sendai-sensitized spleen cells. The relevance of these cell-mediated immune responses to respiratory tract virus infections is discussed.
The presence in the spleens of unsensitized CBA mice of cells that are spontaneously cytotoxic for Sendai virus-infected L cells was confirmed. This innate cytotoxic activity to virus-infected cells was shown to exhibit some H-2 restriction. Partial identity of only the D end of the H-2 gene complex between the target and effector cells was required to produce cytolysis. Attempts to characterize the kind of cell active in this system indicated that neither the theta antigen nor the surface immunoglobulin markers were present. Furthermore, the cells appeared to have no adherent or phagocytic properties. The relationship between the effector cells responsible for innate cytotoxicity to virus-infected cells and the natural killer (NK) cells spontaneously cytotoxic for certain tumor cells is discussed.
Studies of a mouse model of genetic resistance to herpes simplex virus type 1 (HSV-1) indicate that the marrow-dependent effector cell of allogeneic resistance plays an important role in natural resistance to this virus infection. Since the marrow-dependent effector cell appears to be closely related to the natural killer (NK) cells, an NK assay with HSV-1-infected fibroblasts [NK(HSV-1)] has been developed to study this resistance mechanism in humans. Incubation of effector and target cells for 12 to 14 h gave the greatest percent specific release (%SR) and kept spontaneous (51)Cr release from infected target cells below 35%. Patients with Bruton's agammaglobulinemia demonstrated significant kill indicating antiviral antibody was not necessary. Seropositive individuals gave a 9% greater%SR than seronegative individuals. Depletion of B-cells consistently diminished NK (HSV-1) for seropositive individuals and augmented kill for seronegative individuals. Although antiviral antibody produced in culture may contribute to NK (HSV-1), depletion of B-cells allowed quantitation of NK (HSV-1) to the exclusion of most of the antibody-dependent kill. The NK cells detected by this assay showed many of the properties reported for NK cells with K562 targets. Two patients with severe herpesvirus infections demonstrated NK (HSV-1) responses greater than 2 standard deviations below the normal mean. Since normal individuals with virus infections have higher rather than lower natural kill, the low NK (HSV-1) may reflect their susceptibility to the virus infection.
Mouse effector cells mediating natural cell-mediated cytotoxicity against tumor cells were found to contain a low density of Thy 1 antigen. Treatment of nude spleen cells, or spleen cells from mice in which natural reactivity was boosted, with anti-Thy 1.2 plus complement resulted in a substantial decrease in cytotoxicity. The spontaneous cytotoxic reactivity of young, thymus-bearing mice was resistant to such treatment, but repeated exposure to anti-Thy 1.2 plus complement did cause a decrease in lytic activity. By use of congenic anti-Thy 1.2 and effector cells from mice congenic for Thy 1, the effects of the treatment were shown to be specific for Thy 1.2 antigen.
Two types of host reactivities not requiring immunization in the mouse and not mediated by T lymphocytes were compared: resistance of irradiated and nonirradiated F1 hybrids to accept parental grafts of normal or malignant hemopoietic cells (Hh system), and the natural killer cell activity against mouse lymphomas (NK system). The effects of six independent variables known to influence resistance to marrow grafts were investigated in the NK system using YAC-1 lymphoma cells as targets. The following properties were shared: (a) maturation during the fourth week of life; (b) low sensitivity to acute total body irradiation; (c) dependence on the integrity of bone marrow as demonstrated by reduced reactivity in ⁸⁹Sr-treated mice; (d) suppression by a single injection of rabbit anti-mouse bone marrow serum; (e) suppression by a single injection of the anti-macrophage agents silica and i-carrageenan; and (f) suppression by multiple injections of parental spleen cells into F1 mice. These positive correlations are particularly significant because most of the variables have either opposing or no effect on conventional immunity. F1 mice rendered specifically unresponsive to parental marrow grafts, could retain NK cell activity, and genetically susceptible mice could be rendered hyporeactive in terms of NK cells, indicating that the specificities of YAC-1 and Hh-1 incompatible targets were different.
NATURAL KILLER (NK) cells constitute a distinct subgroup of cells within the immune system1,2. They are found in the lymphoid organs of several species including man and are cytolytic on contact in short-term in vitro assays for several cell types, in particular tumour cells1-5. Although the level of NK cell activity is under genetic control6,7, several extraneous agents, including bacterial adjuvants, animal viruses and NK-sensitive tumour cells induce an increase in in vivo NK cell activity6-10. Several of these agents are also known to increase the resistance of mice to transplantable tumours. This effect may be mediated by NK cells, as a positive correlation exists between in vivo resistance to syngeneic tumours and the levels of NK cell activity in the individual mice11,12. Viruses as well as several immunoadjuvants are also inducers of interferon. Here we present evidence that interferon and interferon inducers markedly enhance NK cell activity in mice, and suggest that interferon may be the mediator by which many different agents increase NK cell activity in vivo.
Normal mice contain cytolytic cells with specificity for in vitro grown mouse Moloney leukemia cells. Such killer cells are most frequent in the spleens; lymph node and bone marrow contain less and thymus virtually no killer activity. Peak activity is found around one to three months of age. Spleen cells from genetically athymic mice are as active killer cells as those from normal mice of the same strain. Treatment with anti-theta serum plus complement followed by removal of adherent and surface Ig positive cells by filtration through anti-Ig columns will leave between 1-5% of the original spleen cell population from a normal mouse. These cells have the morphology of small lymphocytes and perhaps contain all of the total original killer activity of the spleen against the Moloney leukemia cells. Such killer enriched cells are devoid of T and B lymphocytes and largely fail to function in antibody induced, cell-mediated lysis against antibody-coated chicken erythrocytes. It is concluded that the spontaneous selective cytotoxic activity of normal mouse spleen cells against Moloney leukemia cells is exerted by small lymphocytes of yet undefined nature.
Spleen cells from untreated young male and female C57BL/6 and C58 mice and of male C3H/He mice showed cytotoxic activity in vitro against BALB/c X-radiation-induced leukemia RL male 1 cells by 51Cr-releasing lymphocyte-mediated cytoxicity (LMC) tests, but old mice of these strains lacked LMC activity. In contrast, spleen cells from male and female AKR, BALB/c, and DBA/2 mice, and from female C3H/He mice had no appreciable LMC activity. The proportion of active cells in spleens from young (C57BL/6 times BALB/c)F1 or reciprocal hybrid mice was higher in females than in males. The specificity of the LMC reaction of RL male 1 cells, determined by LMC inhibition assays, was somewhat different from that of previously reported serologic X.1 tests. Thus the antigen detected by LMC has been tentatively designated X.1'. The main effector cells in this system were uncharacterized cells not adherent to glass surfaces or nylon-wool columns. These findings in RL male 1 leukemia extend the evidence for the presence of naturally occurring LMC. With the single unexplained exception of strain C3H/He, the LMC activity against RL male 1 cells, exhibited by untreated mice of various strains, corresponded with a previous classification of mouse strains immunologically as X.1 responders or as X.1 nonresponders according to their ability to reject X.1-positive leukemia cells.
In the spleens of young, adult mice there exist naturally occurring killer lymphocytes with specificity for mouse Moloney leukemia cells. The lytic activity was directed against syngeneic or allogeneic Moloney leukemia cells to a similar extent, but was primarily expressed when tested against in vitro grown leukemia cells. Two leukemias of non-Moloney origin were resistant and so was the mastocytoma line P815. Although killer activity varied between different strains of mice, the specificity of lysis was the same as indicated by competition experiments using unlabeled Moloney or other tumor cells as inhibitors in the cytotoxic assays. Capacity to compete and sensitivy to lysis by the killer cells were found to be highly positively correlated. Analysis of the kinetics of the cytotoxic assay revealed a rapid induction of lysis within one to four hours, arguing against any conventional in vitro induction of immune response. No evidence was found of soluble factors playing any role in the cytolytic assay.
We investigated a broad spectrum of immunoactive mediators in a mouse model of influenza. ICR mice (4-5 wk old) that were infected with a 10 LD50 dose of influenza A/PR8/34 virus died after 6 days without evidence of bacterial superinfection. Maximal virus titers were reached by day 2 postinfection, whereas the multifocal pneumonia with mononuclear cell infiltration reached its maximum at the end of infection. We measured the cytokines IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-6, IFN-gamma, TNF-alpha, granulocyte (G)/macrophage (M)-CSF, G-CSF, M-CSF, and the lipid mediators leukotriene B4 and platelet-activating factor in the cellfree bronchoalveolar lavage fluid of mice during infection. We found an early increase of IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, GM-CSF, IFN-gamma, and leukotriene B4. Levels of these factors peaked between 36 h and day 3 postinfection, with the exception of IL-6 that remained at elevated levels throughout infection. G-CSF and M-CSF increased slowly and reached a maximum by day 5 postinfection. We were unable to detect IL-2, IL-3, or IL-4. PAF remained at the same level throughout infection. Our results suggest that lung-resident cells, and possibly the alveolar macrophages, participate actively in the onset of the inflammatory response against the invading virus. The inability to detect the T cell products IL-2, IL-3, and IL-4 was unexpected considering the role of T cells in the elimination of the virus in infected mice. Our observation confirms thus earlier findings about the inability of specific T cell clones to elicit an unspecific antiviral effect.
The V(D)J recombination activation gene RAG-1 was isolated on the basis of its ability to activate V(D)J recombination on an artificial substrate in fibroblasts. This property and the expression pattern in tissues and cell lines indicate that RAG-1 either activates or catalyzes the V(D)J recombination reaction of immunoglobulin and T cell receptor genes. We here describe the introduction of a mutation in RAG-1 into the germline of mice via gene targeting in embryonic stem cells. RAG-1-deficient mice have small lymphoid organs that do not contain mature B and T lymphocytes. The arrest of B and T cell differentiation occurs at an early stage and correlates with the inability to perform V(D)J recombination. The immune system of the RAG-1 mutant mice can be described as that of nonleaky scid mice. Although RAG-1 expression has been reported in the central nervous system of the mouse, no obvious neuroanatomical or behavioral abnormalities have been found in the RAG-1-deficient mice.
We have generated mice that carry a germline mutation in which a large portion of the RAG-2 coding region is deleted. Homozygous mutants are viable but fail to produce mature B or T lymphocytes. Very immature lymphoid cells were present in primary lymphoid organs of mutant animals as defined by surface marker analyses and Abelson murine leukemia virus (A-MuLV) transformation assays. However, these cells did not rearrange their immunoglobulin or T cell receptor loci. Lack of V(D)J recombination activity in mutant pre-B cell lines could be restored by introduction of a functional RAG-2 expression vector. Therefore, loss of RAG-2 function in vivo results in total inability to initiate V(D)J rearrangement, leading to a novel severe combined immune deficient (SCID) phenotype. Because the SCID phenotype was the only obvious abnormality detected in RAG-2 mutant mice, RAG-2 function and V(D)J recombinase activity, per se, are not required for development of cells other than lymphocytes.
CD4-, CD8- thymocytes were purified from thymi obtained from normal C57BL/6 mice. By flow cytometry analysis, 5 to 10% of these double negative (DN) thymocytes were found to express NK1.1 on their surface. The NK1.1+ DN thymocytes were demonstrated, by two-color fluorescence, to be CD3lo, CD5hi, CD44hi, J11d-, B220-, MEL 14-, IL2R- with 60% expressing TCR-V beta 8 as determined by the mAb F23.1. In contrast, splenic and peripheral blood NK cells were NK1.1+, CD3-, CD5-, TCR-V beta 8- with 40 to 60% being MEL 14+. Unlike peripheral NK cells, fresh DN thymocytes enriched for NK1.1+ cells were unable to kill YAC-1, the classical murine NK cell target. However, these cells were able to mediate anti-CD3 redirected lysis even when they were assayed immediately after purification, i.e., with no culture or stimulation. These data demonstrate that adult murine thymocytes contain NK1.1+ cells which are distinct, both by function and phenotype, from peripheral NK cells. These data also raise the issue of a possible NK/T bipotential progenitor cell.
Ly-49 (YE1/48, A1) is a dimer protein expressed on subpopulations of murine NK cells. It is a member of a superfamily of type II transmembrane proteins containing carbohydrate recognition domains (CRD). In the mouse genome, the detection of multiple restriction fragments that cross-hybridize with Ly-49 cDNA probes suggests the presence of related genes. In this study, we have isolated several genomic clones encoding portions of CRD sequences highly homologous to the CRD of Ly-49. By using primers based on the consensus sequences of the genomic clones, expression of Ly-49-related genes was detected by the polymerase chain reaction in various organs, including lung, kidney, liver, spleen, and thymus. Two full-length cDNA clones that are highly homologous to the Ly-49 gene were subsequently isolated from a lung cDNA library. At the nucleotide level, the two clones are 72% and 80% identical to Ly-49 in their translated regions, but their sequences are different from those of the genomic clones characterized to date. The two cDNA clones potentially encode type II transmembrane proteins containing CRD that are very similar to Ly-49. These amino acid sequences are also homologous to other members of the superfamily of CRD-containing type II transmembrane proteins, including hepatic lectins and the low affinity IgER (CD23). The homology is most evident in the CRD but is also significant in other domains. These results demonstrate the existence of several functional genes that are highly related to Ly-49. These genes comprise a subfamily within the superfamily of type II transmembrane proteins containing CRD.
The Ly-49 (A1, YE1/48) Ag is a disulfide-linked dimer with 44-kDa subunits, and is expressed on the cell surface of rare T cell tumors of C57BL/6 origin. Although this Ag is undetectable by flow microfluorimetry analysis, normal cells have been shown to express the A1 Ag by immunoprecipitation experiments performed on surface radioiodinated spleen and thymus cells. We (J. Immunol. 143:1379, 1989) and others (J. Immunol. 142:1727, 1989) have recently isolated cDNA encoding the Ly-49 Ag. Southern blots with an Ly-49 cDNA probe revealed multiple bands, consistent with cross-hybridization to other members of a multigene family, and significant RFLP between the C57BL/6 and BALB/c strains. When the RFLP patterns displayed by other common laboratory strains as well as informative recombinant inbred strains were examined, the Ly-49 gene family displayed five RFLP patterns and the entire family was found to reside on a contiguous stretch of the distal portion of mouse chromosome 6, the same region to which the NK1.1 Ag has been mapped. Although tissue distribution studies and transfection analysis ruled out the possibility that Ly-49 was identical to NK1.1 Ag, approximately 20% of NK1.1 cells isolated from normal spleen coexpressed Ly-49 and all Ly-49+ cells were CD3-. Although spleen cells cultured in high doses of rIL-2 demonstrated similar coexpression of NK1.1 and Ly-49, approximately 10% of CD3+ cells coexpressed Ly-49. The chromosomal mapping data and the expression of the Ly-49 and NK1.1 Ag suggest that the NK1.1 Ag may be a member of the Ly-49 multigene family.
Influenza and pneumonia are leading causes of death in the elderly. Cytotoxic T-lymphocyte activity is responsible for viral clearance after infection and declines with age. We hypothesized that following intranasal infection with influenza virus, aged mice would have decreased anti-influenza cytotoxic T-lymphocyte activity that would correlate with prolonged pulmonary viral shedding. To test this, young (1.5-4.0 month) and aged (22-25 month) BALB/c mice were infected intranasally with influenza A/Port Chalmers/1/73(H3N2). Mice were killed at 3-19 days following infection. Their splenic cytotoxic T-lymphocyte activity was measured by a secondary in vitro chromium release assay. Pulmonary viral titres were quantified by growth of titrated lung specimens in fertilized hens' eggs. Serum antibody titres were measured by an ELISA. Young mice responded in a relatively homogeneous fashion. They developed maximal cytotoxic T-lymphocyte activity of 60.9 +/- 2.0% by Days 11-13, and all except one cleared virus from the lung by Day 7. In contrast, old mice were heterogeneous. Their cytotoxic T-lymphocyte activity peaked at 46.9 +/- 5.0% and was delayed by 5-7 days. Forty-five per cent were still shedding virus at Days 7 and 8, and shedding persisted for at least 13 days in some mice. There was a strong correlation in both young and aged mice between the presence of virus in the lungs and decreased splenic cytotoxic T-lymphocyte activity (chi 2 = 30.2, P much less than 0.001). No significant difference was found between young and aged animals in serum IgG1 anti-H3 antibody titres. We conclude that following influenza infection in aged mice, impaired cytotoxic T-lymphocyte activity leads to prolonged duration of infection. These observations may lead to a better understanding of the excess morbidity and mortality in elderly persons that occur with influenza.
Why does influenza virus variously eventuate in mild upper respiratory tract infection, bacterial pneumonia, or rapidly fatal viral pneumonia? The depth of infection is important. So is competence of immunologic effectors--local antibody (secretory IgA) for preventing URI, systemic antibody for preventing pneumonia, and cellular immunity for recovery from either.
We have previously reported on the antimetastatic effects of experimental adoptive immunotherapy using plastic adherent lymphokine-activated killer cells (A-LAK) cells (R. E. Schwarz et al. Cancer Res. 49: 1441, 1989). We have also reported that the spleen is a superior source of lymphocytes for A-LAK cell generation (R. E. Schwarz and J. C. Hiserodt, Med. Hypotheses 28: 165, 1989). This study, therefore, was designed to examine the effects of splenectomy itself on tumor growth in an experimental animal model. Natural killer (NK)-resistant MADB106 mammary adenocarcinoma cells were injected iv into F344 rats to generate multiple lung metastases. Splenectomies (Sx) were performed on Days -6, -3, -1, 0, 1, 3, 6, and 10, counted from the time of tumor injection. Groups consisted of six animals each, and sham-anesthetized and -operated animals served as controls. Splenectomies, if performed between Days -3 and +1, had significant antitumor effects as documented by the number of outgrowing surface metastases (5 +/- 7 vs greater than 300; P less than 0.0001) and by animal survival (greater than 100 vs 21 +/- 3 days; P less than 0.001). However, splenectomies, performed at an earlier or later stage, did not show these effects. Sx did not alter peripheral blood NK activity or the percentages of mononuclear cell subsets except for a slight decrease in the T-helper/T-suppressor ratio (P less than 0.04). Interleukin 2 (rhIL2), given at 2.5 X 10(5) U/kg/day for 3 days immediately after splenectomy, completely abrogated the observed antitumor effects. Subcutaneous tumor rechallenge of long-term surviving animals showed no tumor take in 87% of the animals.(ABSTRACT TRUNCATED AT 250 WORDS)