A detrimental role for invariant natural killer T cells in the pathogenesis of experimental dengue virus infection

Dengue virus (DENV), a member of the mosquito-borne flaviviruses, is a serious public health problem in many tropical countries. We assessed the in vivo physiologic contribution of invariant natural killer T (iNKT) cells, a population of nonconventional lipid-reactive αβ T lymphocytes, to the host response during experimental DENV infection. We used a mouse-adapted DENV serotype 2 strain that causes a disease that resembles severe dengue in humans. On DENV challenge, splenic and hepatic iNKT cells became activated insofar as CD69 and Fas ligand up-regulation and interferon-γ production. C57BL/6 mice deficient in iNKT cells (Jα18(-/-)) were more resistant to lethal infection than were wild-type animals, and the phenotype was reversed by adoptive transfer of iNKT cells to Jα18(-/-) animals. The absence of iNKT cells in Jα18(-/-) mice was associated with decreased systemic and local inflammatory responses, less liver injury, diminished vascular leak syndrome, and reduced activation of natural killer cells and neutrophils. iNKT cell functions were not necessary for control of primary DENV infection, after either natural endogenous activation or exogenous activation with the canonical iNKT cell agonist α-galactosylceramide. Together, these data reveal a novel and critical role for iNKT cells in the pathogenesis of severe experimental dengue disease.     

Antibodies play a greater role than immune cells in heterologous protection against secondary dengue virus infection in a mouse model

The four serotypes of dengue virus (DENV1–4) are causative agents of dengue fever and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Previous DENV infection is a risk factor for DHF/DSS during subsequent infection by a different serotype. Nonetheless, most primary and secondary DENV infections are asymptomatic. To investigate the possible mechanisms of immune protection in vivo, 129/Pas mice lacking IFN-α/β and -γ receptors (AG129) were used to model secondary infection using both DENV1–DENV2 and DENV2–DENV4 sequences. At intervals between sequential infections of 4 to 52 weeks, protection against secondary heterologous DENV infection was observed. Passive transfer of DENV-immune serum was protective against replication of heterologous challenge virus in all tissues tested, whereas adoptive transfer of DENV-immune cells significantly protected mice from replication of the challenge virus only when a lower inoculum was administered. These findings are relevant for understanding both natural and vaccine-induced immunity to DENV.     

CD4+ cells play a limited role in murine lung infection with Mycobacterium kansasii

Mycobacterium kansasii has emerged as an important nontuberculous mycobacterium that can cause severe infection in the immunocompromised host, especially in human immunodeficiency virus-infected patients. However, little is known about the pathogenesis of this infection. Because patients suffering from M. kansasii infection are severely compromised in their cellular immune response, we studied the course of infection in CD4+ cell knockout (KO) mice. Wild-type (WT) mice and CD4+ KO mice were infected with 10(5) cfu of M. kansasii. Although previously shown to be susceptible to Mycobacterium tuberculosis infection, CD4+ KO mice demonstrated no impairment in clearing infection with M. kansasii when compared with WT animals, despite reduced pulmonary inflammation (reduced granuloma formation and lymphocyte infiltration in the lungs). Pulmonary IFN-gamma levels and M. kansasii-induced IFN-gamma production by splenocytes from infected animals were reduced in CD4+ KO mice, confirming that these mice were defective in the M. kansasii-specific T helper cell type 1 immune response. Furthermore, mice deficient for IFN-gamma, IL-12p35, IL-12p40, or IL-18 also displayed a normal host defense against pulmonary infection with M. kansasii. These data suggest that CD4+ cells, IFN-gamma, and an intact T helper cell type 1 response play a limited role in protective immunity against pulmonary M. kansasii infection.     

Early activation of natural killer and B cells in response to primary dengue virus infection in A/J mice

Dengue virus (DEN) causes the most prevalent arthropod-borne viral illness in humans worldwide. Immune mechanisms that are involved in protection and pathogenesis of DEN infection have not been fully elucidated due largely to the lack of an adequate animal model. Therefore, as a first step, we characterized the primary immune response in immunocompetent inbred A/J mice that were infected intravenously with a non-mouse-adapted DEN type 2 (DEN2) strain. A subset (55%) of infected mice developed paralysis by 14 days post-infection (p.i.), harbored infectious DEN in the central nervous system (CNS), and had an elevated hematocrit and a decreased white blood cell (WBC) count. Immunologic studies detected (i). increased numbers of CD69(+) splenic natural killer (NK) and B cells at day 3 p.i., (ii). DEN-specific IgM and IgG responses by days 3 and 7 p.i., respectively, and (iii). splenocyte production of IFNgamma at day 14 p.i. We conclude that the early activities of NK cells, B cells and IgM, and later actions of IFNgamma and IgG likely play a role in the defense against DEN infection.     

Endogenous CD4(+) CD25(+) regulatory T cells have a limited role in the control of Trypanosoma cruzi infection in mice

Infection with the protozoan parasite Trypanosoma cruzi results in a robust and multifaceted immune response that controls parasite load but is unable to completely clear infection, resulting in parasite persistence and a chronic illness known as Chagas' disease in humans. The severity of Chagas' disease is correlated with persistent parasitism of muscle, neuronal, and gut tissues. The natural immunomodulatory function of endogenous CD4(+) CD25(+) regulatory T cells (Treg cells) to limit hyperactive immune responses may be exploited by microbes to persist despite host responses. In this study, we show that Treg cells are not necessary for T. cruzi evasion of immune responses during acute or chronic infection. In vivo anti-CD25 monoclonal antibody-mediated depletion of Treg cells from mice prior to challenge with a lethal strain or prior to and during acute infection with a nonlethal strain of parasite neither improved nor worsened the outcome of immune responses: differences in parasitemia, kinetics of antigen-specific CD8(+) T-cell expansion, and CD8(+) T-cell effector function (both in vivo and ex vivo) were of similar magnitudes for both depleted and control groups. Furthermore, depletion of CD25(+) cells from chronically infected mice did not enhance immune responses of muscle-derived CD8(+) T cells, nor could FoxP3 mRNA/scurfin-expressing leukocytes be isolated from muscle tissue. Based on the results of this study, it is concluded that Treg cells do not appear to play a major role in regulating CD8(+) T-cell effector responses during the acute phase of infection or in the muscles of mice during chronic T. cruzi infection.     

Functional role of phosphatidylcholine-specific phospholipase C in regulating CD16 membrane expression in natural killer cells

CD16, the low-affinity FcIgG receptor (FcgammaRIIIA), is predominantly expressed in human NK cells. Our recent findings indicate that CD16 expression on the outer membrane surface of NK cells is correlated with the membrane expression of phosphatidylcholine-specific phospholipase C (PC-PLC). In the present study we analyzed the trafficking of CD16 from the plasma membrane to cytoplasmic regions, after stimulation with specific mAb. The CD16 receptor is internalized, likely degraded and newly synthesized; its endocytosis is independent of ATP, but requires an integral and functional actin cytoskeleton. Antibody-mediated CD16 cross-linking results in an approximately twofold increase in PC-PLC enzymatic activity within 10 min. Analysis of PC-PLC and CD16 distribution in NK cell plasma membrane demonstrates that the proteins are physically associated and partially accumulated in lipid rafts. Pre-incubation of NK cells with a PC-PLC inhibitor, D609, causes a dramatic decrease both in CD16 receptor and PC-PLC enzyme expression on the plasma membrane. Interestingly, among phenotype PBL markers, only CD16 is strongly down-modulated by D609 treatment. CD16-mediated cytotoxicity is also reduced after D609 incubation. Taken together, these data suggest that the PC-PLC enzyme could play an important role in regulating CD16 membrane expression, the CD16-mediated cytolytic mechanism and CD16-triggered signal transduction.     

CD14 plays a limited role during influenza A virus infection in vivo

Influenza A is a single stranded (ss)RNA virus that can cause upper respiratory tract infections that in rare cases may progress to pneumonia. Toll-like receptors (TLRs) and CD14 are receptors which recognize viral proteins and nucleic acid of several viruses. CD14 is required for influenza-induced cytokine production during infection of mouse macrophages. In addition, CD14 was shown to bind ssRNA, suggesting an important role for CD14 during infection with influenza. To investigate the role of CD14 during influenza pneumonia we inoculated WT and CD14 KO mice with a non-lethal dose of a mouse adapted strain of influenza A. CD14 KO mice displayed a reduced viral load in the lungs, 2 and 14 days after infection with influenza. Pulmonary cytokine production in CD14 KO mice was reduced at day 2 and elevated at day 8 compared to WT mice. CD14 deficiency did not influence lymphocyte recruitment or lymphocyte activation in lungs and draining lymph nodes 8 days after infection. These data show that CD14 plays a limited role in host defense against infection with influenza.     

Double-edged role of natural killer cells during RSV infection

Abstract

Natural killer cells play a vital role in the rejection of tumors and pathogen-infected cells. NK cells are indispensable in the early immune response against viral infections by directly targeting infected cells. Furthermore, NK cells influence adaptive immunity by driving virus-specific T-cell responses. Respiratory syncytial virus, a highly contagious virus that causes bronchiolitis, is the main reason for mortality in infants and elderly patients. RSV infection triggers both innate and adaptive immune responses. However, immunity against RSV is ephemeral due to the impaired development of immunological memory. The role of NK cells during RSV infection remains ambiguous. NK cells play a dual role in RSV infection; initially, their role is a protective one as they utilize their intrinsic cytotoxicity, followed by a detrimental one that induces lung injury due to the inhibition of antibody responses and the secretion of pro-inflammatory factors. Noteworthy, IFN-γ released from NK cells play a critical role in promoting a shift to adaptive responses and inhibiting antibody responses in neonates. Indeed, NK cells have a pro-inflammatory and inhibitory role rather than a cytotoxic one that contributes to the severity of the disease. Therapeutic options, including DNA-protein-based vaccines, synthetic peptides, and attenuated strains, are presently under tests. However, there is a need for effective strategies to augment NK cell activity and circumvent the pro-inflammatory activity to benefit the host. In this review, we focused on the role played by NK cells in the immune response and its outcome on the immunopathogenesis of RSV disease.     

CD1d-restricted natural killer T cells are potent targets for human immunodeficiency virus infection

Invariant human natural killer T cells (NKT) express a restricted T-cell receptor (TCR) Valpha24Vbeta11 repertoire. These cells share both phenotypic and functional similarities between NK and T cells. Given the emerging role of NKT cells as critical cells in bridging the gap between innate and adaptive immunity, we examined their susceptibility to productive human immunodeficiency virus (HIV) infection by T-tropic, M-tropic, and primary isolates of HIV. We generated three human NKT cell clones (CA5, CA29, and CA31). Phenotypic characterization of these Valpha24+ Vbeta11+ clones indicated that they were predominately positive for CD4, CD161, HLA-DR, CD38, CD45RO, and CD95 expression. The NKT cell clones expressed significantly more surface CCR5 molecules/cell and lower CXCR4 molecules/cell than phytohaemagglutinin-stimulated peripheral blood mononuclear cells (PBMC). Consistent with the surface expression of CCR5 and CXCR4, the NKT clones were also selectively susceptible to HIV M-tropic, T-tropic, and primary isolate infection, as evaluated by both HIV p24 enzyme-linked immunosorbent assay and intracellular staining of HIV proteins. The amount of p24 production was dependent on the NKT clone studied and the HIV strain used. Clones CA29 and CA31 were also susceptible to HIV IIIB infection. The virions produced by these clones were able to productively infect PHA-stimulated PBMCs with the same kinetics as for primary infection of CD4+ blast. Collectively, this data demonstrates that NKT cells can be a target for productive HIV infection but with a lag in the time to peak p24 production.     

Activation and role of natural killer cells in virus infections

Natural killer cells may play a significant role in virus infections. Virus-induced interferon activates these cells to become highly cytotoxic, and viral infections may also affect the proliferation of such cells. Non-immune mice have an apparently cellular defense mechanism which rapidly lyses implanted virus-infected cells. The evidence for NK cell involvement in viral disease is reviewed.Presented at the Fifth International congress of Virology, Strasbourg, France, 1981.     

CD4(+) v(alpha)14 NKT cells play a crucial role in an early stage of protective immunity against infection with Leishmania major

The roles of gamma delta T, NK and NKT cells in an early stage of protective immunity against infection with Leishmania major were investigated. Further, the contribution of these innate cells to the expression of 65 kDa heat shock protein (HSP65) in host macrophages was examined, since we found previously that this expression prevents apoptotic death of infected macrophages and is a crucial step in the acquisition of protective immunity against infection with various obligate intracellular protozoa including L. major. C57BL/6 and DBA/2 mice were found to be resistant against the infection on the basis of the parasite burden in their regional lymph nodes, and to strongly express HSP65 in their macrophages, whereas BALB/c mice were susceptible and barely expressed the HSP65. In those resistant mice, CD4(+) NKT cells prominently increased in their regional lymph node and were the main effector cells at least for an early stage of the protective immunity and for the HSP65 expression, whereas this subset did not increase in susceptible BALB/c mice. Further, neither gamma delta T nor NK cells in resistant mice contributed to those protective immune responses. The NKT cell subset bore CD3, CD4, TCR alpha beta, IL-2R beta and NK1.1 but scarcely asialo-GM(1). Moreover, this effector subset was confirmed to be V(alpha)14 NKT cells by using J(alpha)281(-/-) mice.     

Natural killer cells appear to play no role in the recovery of mice from Sindbis virus infection.

Previous studies have suggested that non-specific defence mechanisms may be important in the development of age-dependent resistance to fatal Sindbis-virus infection and in the recovery of adult mice from non-fatal infection. In these studies, natural killer (NK) cell induction was studied in 7-day-old susceptible mice and 28-35-day-old resistant mice. It was found that Sindbis virus infection induced NK cells in both the young and older mice, suggesting that NK cells were not important in the acquisition of resistance to fatal Sindbis-virus infection. Transfer of 10(8) lymph node cells from adult, mice, at the peak of NK cell activity, did not protect young mice from fatal infections, supporting the in vitro findings. The pathogenesis of Sindbis virus infection in C57BL/6J bg/bg (NK-cell deficient) and bg/+ (NK-cell normal) mice was also studied. Despite a defect in the induction of NK cells by Sindbis virus infection in the bg/bg mice, there were no significant differences in the pathogenesis of either peripheral or intracerebral infection in these strains of mice. These studies suggest that although NK cells are induced, they may not be important in the recovery of mice from Sindbis virus infection.     

Defining the role of NK cells during dengue virus infection

In recent years, our understanding of the complex number of signals that need to be integrated between a diverse number of receptors present on natural killer (NK) cells and ligands present on target cells has improved. Here, we review the progress made in identifying interactions between dengue viral peptides presented on HLA Class 1 molecules with inhibitory and activating killer‐like immunoglobulin receptors on NK cells, direct interactions of viral proteins with NK cell receptors, the involvement of dengue virus‐specific antibodies in mediating antibody‐dependent cell‐mediated cytotoxicity and the role of soluble factors in modulating NK cell responses. We discuss findings of NK cell activation early after natural dengue infection, and point to the role that NK cells may play in regulating both innate and adaptive immune responses, in the context of our new appreciation of interactions of dengue virus with specific NK cell receptors. With a number of flavivirus vaccine candidates in clinical trials, how NK cells respond to attenuated dengue virus and subunit protein vaccine candidates and shape adaptive immunity will need to be considered.     

CD4(+) T helper cells and the role they play in viral control

The natural history of untreated HIV-1 infection is characterized by progressive erosion of the immune system with eventual loss of viral control. T helper cell responses to HIV-1 are typically weak or absent in chronically HIV-1 infected individuals. CD4(+) T helper cell responses have been shown to be important in a number of experimental viral infection systems, and here we explore the ways in which T helper cells might aid in the control of viruses in general and HIV-1 in particular. We first review the relationship between T helper cells and other arms of the immune system. We then focus on the role T helper cells play in viral control in murine and nonhuman primate models, and finally review what is known of CD4(+) T helper cell function in HIV-1 infection.     

Abundance of unconventional CD8(+) natural killer T cells in the large intestine.

Natural killer T (NKT) cells are mainly present in the liver and thymus, and the majority of these T cells express either a CD4(+) or a double-negative (DN) CD4(-)8(-) phenotype. In the present study, we examined whether such NKT cells were present in the intestine. NKT cells were rare in all sites of the small intestine, including an intraepithelial site. However, a considerable number of NKT cells were found at an intraepithelial site in the large intestine. This result was confirmed by both immunofluorescence and immunohistochemistry. In contrast to conventional NKT cells, NKT cells in the large intestine were CD8(+) or DN CD4(-)8(-). In the case of conventional NKT cells, their existence is known to depend on non-classical MHC class I-like antigens (i. e. CD1d) but not on classical MHC class I antigens. However, the NKT cells in the large intestine were independent of the presence of both CD1d and classical MHC class I antigens. These results were obtained using knockout mice lacking the corresponding genes and molecules. NKT cells in the large intestine were mainly alpha betaTCR(+) (> 75 %) but did not use an invariant chain of Valpha14Jalpha281, which is preferentially used by conventional NKT cells. These NKT cells did not bias the TCR-Vbeta usage toward Vbeta8. These findings suggest that the large intestine is a site in which unconventional NKT cells carrying the CD8(+) phenotype (or DN CD4(-)8(-)) are abundant and that these cells are independent of MHC and MHC-like antigens.     

Early-appearing tumour-infiltrating natural killer cells play a crucial role in the generation of anti-tumour T lymphocytes.

Natural killer (NK) cells that infiltrated into the primary tumour site at an early stage of tumour development, were examined for their participation in the generation of anti-tumour cytotoxic T lymphocytes (CTL). NK cells, which were detected by anti-NK1.1 monoclonal antibody (mAb), increased in the peritoneal exudate cells (PEC) on days 3 and 7 after an intraperitoneal (i.p.) inoculation of syngeneic B16 melanoma cells. These tumour-infiltrating NK cells showed a high level of cytotoxic activity against NK-sensitive YAC-1 cells and an increased expression of interferon-gamma (IFN-gamma) mRNA and interleukin-2 (IL-2) mRNA. The in vivo depletion of NK cells with anti-NK1.1 mAb, prior to i.p. inoculation of B16 melanoma cells, resulted in an increased number of tumour cells in the PEC compared to NK cell non-depleted mice. Interestingly, the differences in tumour cell number between both groups were more prominent on days 7 and 14 than on day 3, which strongly suggested that early-infiltrating NK cells have a large influence on the subsequent anti-tumour response. The in vivo depletion of NK cells prior to immunization with melanoma cells abrogated the capacity of the spleen cells to generate CD8+ tumour-specific CTL after in vitro restimulation. This inability of generating anti-tumour CTL was partially restored by additional i.p. injections of recombinant IL-2 and/or IFN-gamma simultaneously with the immunization of melanoma cells. The in vitro depletion of NK cells prior to the in vitro restimulation with melanoma cells partially impaired the anti-tumour CTL generation from the spleen cells of the immunized mice. Lastly, the in vivo depletion of NK cells prior to immunization with melanoma cells abolished the protective immunity against melanoma cells at the rechallenge. Overall, these results indicate that early-appearing tumour-infiltrating NK cells not only participate in the anti-tumour early defence by themselves, but also play a crucial role in the generation of anti-tumour CTL.     

A novel mutation in the FcgammaRIIIA gene (CD16) results in active natural killer cells lacking CD16

We report a novel mutation in FcgammaRIIIA (the transmembrane-form CD16) on natural killer (NK) cells in a patient with polyneuropathy. She had no history of recurrent infections. Her NK cells expressed no detectable CD16; however, her NK cytotoxic activity was normal, suggesting that CD16 expression and cytotoxic activity are independent of one another. Molecular analysis revealed a deletion of a single adenine base in exon 4 of CD16 at nucleotide 550. This deletion generates a STOP codon in an extra-cellular domain of the FcgammaRIIIA gene, thereby truncating the CD16 molecule. The patient's NK cells were not recognized by the anti-CD16 monoclonal antibodies 3G8 and Leu11c. Whether the development of her polyneuropathy is associated with this novel mutation is unclear.     

A multi-step isolation scheme for obtaining CD16+ human natural killer cells

A multi-step isolation scheme capitalizing on negative selection protocols is described for obtaining an enriched population of CD16+ human natural killer (NK) cells. The isolation scheme consists of incubating peripheral blood mononuclear cells (MNC) on nylon wool, rosetting the nylon wool non-adherent cells with sheep red blood cells (SRBCs) for 1 h at 29 degrees C and then utilizing a 'panning' technique to remove CD3+, non-rosetting cells. The final working cell population contained 70-80% CD16+ cells, 15% CD2+ cells, 1-3% CD3+ cells, 5-7% SIg+ cells and no detectable MO2+ cells. In comparing the final NK cell population from the multi-step isolation protocol to NK cells obtained by the Percoll density gradient centrifugation technique, the multistep method: (1) yielded a higher percentage of CD16+ cells, (2) mediated a greater degree of cytotoxicity at a 25:1 E:T ratio, and (3) contained fewer contaminating monocytes/macrophages (none were detectable). In addition, the multi-step scheme allowed recovery of 30% of the total CD16+ cells present compared to only 7% recovered by the Percoll density gradient technique. Pretreatment of the enriched NK cells, obtained from the multi-step scheme, with interleukin-2 (3.5 and 7.0 U/ml of activity) resulted in an increase in NK cell-mediated cytotoxicity. In addition, these cells were as effective at synthesizing the cytotoxin, NKCF, at a 25:1 E:T ratio as at 50:1 and 100:1 E:T ratios. This multi-step isolation scheme consistently yields a high percentage of CD16+ NK cells and thus may greatly facilitate studies on the mechanism(s) involved in NK cell-mediated cytotoxicity and may further the study of the cytotoxins involved.     

In vivo acute depletion of CD8(+) T cells before murine cytomegalovirus infection upregulated innate antiviral activity of natural killer cells

In this study, we investigated the effect of depletion of CD8(+) T cells on the activity of natural killer (NK) cells at an early phase of murine cytomegalovirus (MCMV) infection. For CD8(+) T cell depletion, mice were intraperitoneally treated with anti-CD8 mAb, purified from 2.43 hybridoma, for 2 consecutive days before or after infection. Three days after infection, we found that an acute depletion of CD8(+) T cells before infection caused a significant decrease in the viral load in liver and spleen. This effect coincided with an increase in numbers of CD3(-) NK1.1(+) cells in spleen and their expression of the early activation molecule CD69. Although cytolytic activity of NK cells increased on day 3 of infection in CD8-depleted mice, the level of IFN-gamma decreased in serum and supernatant of cultured spleen cells. In contrast to the effect of acute depletion of CD8(+) T cells before infection, the depletion after infection had no effect on the viral load or number and cytolytic function of NK cells. Lack of effects of CD8(+) T cell depletion on the viral load and NK cytolytic activity is also observed in CD8(+) knockout mice. In conclusion, the results suggest that an acute depletion of CD8(+) T cells before MCMV infection effectively upregulated the antiviral activity of NK cells. This effect appears to be mediated through an increase in numbers, activation and cytolytic activity of NK cells.