Cellular Innate Immune Responses Research Articles

Vaccination with heat-killed leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4+ and CD8+ T cell responses and protection against leishmania major infection.

CpG oligodeoxynucleotides (ODN) have potent effects on innate and adaptive cellular immune responses. In this report, the ability of CpG ODN to confer long-term immunity and protection when used as a vaccine adjuvant with a clinical grade of leishmanial antigen, autoclaved Leishmania major (ALM), or a recombinant leishmanial protein was studied. In two different mouse models of L. major infection, vaccination with ALM plus CpG ODN was able to control infection and markedly reduce lesion development in susceptible BALB/c and resistant C57BL/6 (B6) mice, respectively, up to 12 wk after immunization. Moreover, B6 mice immunized with ALM plus CpG ODNs were still protected against infectious challenge even 6 mo after vaccination. In terms of immune correlates of protection, ALM plus CpG ODN-vaccinated mice displayed L. major–specific T helper cell 1 and CD8+ responses. In addition, complete protection was markedly abrogated in mice depleted of CD8+ T cells at the time of vaccination. Similarly, mice vaccinated with a recombinant leishmanial protein plus CpG ODN also had long-term protection that was dependent on CD8+ T cells in vivo. Together, these data demonstrate that CpG ODN, when used as a vaccine adjuvant with either a recombinant protein or heat-killed leishmanial antigen, can induce long-term protection against an intracellular infection in a CD8-dependent manner.

Deactivation of the Innate Cellular Immune Response Following Endotoxic and Surgical Injury

The innate cellular immune (iCMI) system provides for the rapid production of interferon-γ (IFNγ) by NK cells in response to microbial threats. In this review, we examine the cellular and cytokine mechanisms of innate cellular immunity as determined in murine endotoxemia. This will be contrasted to the subsequent suppression of these same responses present in the mouse model of endotoxin tolerance, which is characterized by profound deficiency in both IL-12 and IFNγ synthesis. Transient IFNγ deficiency due to altered iCMI function has also been described in trauma or burn patients and is termed “clinical immune paralysis.” If the common pathogenesis of these entities can be better understood, immune-based interventions might be identified for restoring iCMI function. In addition to the gain in basic immunologic insight, research on this subject may deliver future forms of prophylaxis against infection that do not rely on antibiotics and that will not promote antimicrobial resistance.

Oral administration of yeast, Saccharomyces cerevisiae, enhances the cellular innate immune response of gilthead seabream ( Sparus aurata L.)

The effects of including lyophilised whole yeast, Saccharomyces cerevisiae, in the diet on the seabream innate immune response were investigated. Gilthead seabream ( Sparus aurata L.) specimens were fed four different diets for 4 weeks: a commercial diet as control and the same diet supplemented with 1, 5 or 10 g/kg yeast. After 1, 2 and 4 weeks, serum complement titres, as a humoral parameter, and phagocytic, respiratory burst, myeloperoxidase and natural cytotoxic activities of head-kidney leucocytes, as cellular parameters, were evaluated. The results showed that yeast supplements enhanced all the latter responses, but not the humoral response. This enhancement was dose-dependent except for the cytotoxic activity that was only stimulated by the lower dose of yeast assayed. As yeast cell walls are able to enhance the seabream cellular innate immune response, these results support the possible use of whole yeast as natural inmunostimulants in common fish diets.

Development of a Langerhans cell-targeted gene therapy format using a dendritic cell-specific promoter.

Langerhans cells (LC), which are a skin-specific member of the dendritic cell (DC) family of antigen presenting cells, play critical roles in the initiation of cellular immune responses in the skin. We developed a LC-targeted gene therapy format in this study, aimed at the establishment of in situ protocols for genetic manipulation of LC function. Dectin-2 is a unique C-type lectin that is expressed selectively by DC, including epidermal LC. A 3.2 kb 5' flanking fragment isolated from the mouse dectin-2 gene, termed the dectin-2 promoter (pDec2), exhibited significant transcriptional activities in epidermal-derived DC lines of the XS series, but not in any of the tested non-DC lines. When pDec2-driven luciferase gene (pDec2-Luc) or enhanced green fluorescence protein gene (pDec2-EGFP) was delivered to mouse skin using the gene gun, expression of the corresponding gene product was observed in the epidermal compartment almost exclusively by the IA+ population (ie LC). LC in the gene gun-treated sites showed features of mature DC and they migrated to the draining lymph node, suggesting that LC-targeted gene expression may lead to the development of immune responses. In fact, EGFP-specific cellular immune responses became detectable after gene gun-mediated delivery of pDec2-EGFP plasmid. These results introduce a new concept that LC function can be genetically manipulated in situ by the combination of gene gun-mediated DNA delivery and a DC-specific promoter.

Human anti-porcine gammadelta T-cell xenoreactivity is inhibited by human FasL expression on porcine endothelial cells.

The role of gammadelta T cells during an immune response is still elusive and has been proposed to play a first line of defense along with other cells of the innate immune system, such as macrophages and natural killer cells, before alphabeta T-cell activation occurs. Innate cellular immune response plays a major role in xenograft rejection. We investigated the response of human gammadelta T cells to unmodified and human FasL (hFasL)-expressing xenogenic porcine endothelial cells. A 51Cr release assay was used to study the xenoreactivity of human gammadelta T-cell clones against porcine endothelial cells. Stable transfectants of porcine endothelial cells expressing hFasL were established and analyzed for their effectiveness in controlling this response. Of the gammadelta T-cell clones tested, 38.9% were cytotoxic for porcine endothelial target cells. This cytotoxic response of human gammadelta T-cell clones was significantly inhibited by a monoclonal antibody against human CD3. Incubation of gammadelta T-cell clones with concanamycin A, an inhibitor of the perforin/granzyme B pathway, caused inhibition of lysis of porcine endothelial cells. Inhibition was not observed upon incubation with either anti-FasL or anti-tumor necrosis factor-alpha monoclonal antibodies. Expression of hFasL on porcine endothelial cells significantly reduced lysis by human gammadelta T cells. These results imply that human gammadelta T cells may represent an important obstacle to xenotransplantation. Specific strategies targeted at this subset of T cells could be important in controlling innate cellular response to xenografts and facilitate graft survival.

Virally Induced Lytic Cell Death Elicits the Release of Immunogenic GRP94/gp96

Necrotic cell death yields the release of cellular components that can function in the initiation of cellular immune responses. Given the established capacity of the endoplasmic reticulum chaperone GRP94 (gp96) to elicit CD8(+) T cell activation, we have investigated the cellular fate and antigenicity of GRP94 in differing scenarios of cell death. Virally induced cell death or mechanical cell death, elicited by freeze/thaw treatment of cell suspensions, yielded GRP94 release into the extracellular space; apoptotic cell death occurring in response to serum deprivation did not elicit GRP94 release. To assess the antigenicity of GRP94 released following virally induced cell death (lethal infection of cells with rVV ES-OVA(Met258-265), a recombinant, ovalbumin epitope-expressing vaccinia virus) or mechanical cell death (freeze/thaw of ovalbumin-expressing cells), tissue culture supernatant fractions were pulsed onto antigen-presenting cells, and antigen re-presentation was assayed as activation of an ovalbumin-specific T cell hybridoma. For both cell death scenarios, released GRP94 elicited a dose-dependent, ovalbumin-specific, hybridoma activation. In contrast, calreticulin derived from rVV ES-OVA(Met258-265)-infected cell extracts did not stimulate B3Z activity. These data identify GRP94 as an antigenic component released upon pathological, but not apoptotic, cell death and provide an assay system for the identification of cellular components of related activity.

Immunomodulatory effects of dietary intake of chitin on gilthead seabream (Sparus aurata L.) innate immune system

To determine the effects of chitin (poly [1→4]-β-N-acetyl-D-glucosamine) on the innate immune response of gilthead seabream (Sparus aurata L.), fish were fed diets containing 0mg (control), 25, 50 or 100mg kg−1chitin for 2, 4 and 6 weeks. Lysozyme and natural haemolytic complement activities, together with head-kidney leucocyte respiratory burst, phagocytic and cytotoxic activities, were studied at each of the assayed times. Lysozyme activity was unaffected by the administration of chitin. The innate humoral and cellular immune response activities assayed were enhanced by the dietary intake of chitin, each increasing at a different time: natural haemolytic complement activity and cytotoxic activity after 2 weeks of treatment, respiratory burst activity from 4 weeks and phagocytic activity after 6 weeks, although, unlike the other activities, no statistically significant differences were observed in the first. The results indicate that chitin increases the activity of the seabream innate immune system, and its use as an immunostimulant is discussed, especially with regards to the protective role.

Pretreatment with recombinant Flt3 ligand partially protects against progressive cutaneous leishmaniasis in susceptible BALB/c mice.

Dendritic cells are potent antigen-presenting cells that also produce interleukin-12 (IL-12) during innate and adaptive cellular immune responses and that thereby promote the differentiation of gamma interferon (IFN-gamma)-producing Th1-type CD4(+) T lymphocytes. We hypothesized that expanded dendritic-cell populations in mice pretreated with the hematopoietic cytokine Flt3L would protect against cutaneous Leishmania major infection. Pretreatment of disease-susceptible BALB/c mice with 10 microg of recombinant Flt3L (rFlt3L) for 9 to 10 days before infection increased lymph node IL-12 p40 productive capacity 20-fold compared to that of saline-injected controls. Furthermore, 9 of 22 (40.9%) rFlt3L-pretreated BALB/c mice resolved their cutaneous infections, whereas none of the 22 control BALB/c mice healed. Healed, rFlt3L-pretreated mice did not develop disease following reinfection. Flt3L pretreatment also reduced parasite numbers 1,000-fold in the cutaneous lesions at 2 weeks after infection relative to numbers in lesions of untreated controls. However, Flt3L pretreatment did not significantly alter L. major-induced IFN-gamma and IL-4 production in lymph node culture at 1, 2, and 4 weeks after infection. Despite the lack of Th immune deviation, Flt3L ligand-pretreated lymph nodes expressed up to 10-fold higher levels of IL-12 p40 and inducible (type 2) nitric oxide synthase mRNA at 7 days after infection. In contrast, treatment with rFlt3L after infection failed to protect against disease despite comparable expansions of dendritic cells and IL-12 p40 productive capacity in both infected and uninfected BALB/c mice treated with rFlt3L. We conclude that rFlt3L pretreatment before infection with L. major reduces parasite load and promotes healing of cutaneous lesions without stable cytokine deviation towards a dominant Th1 cytokine phenotype.

Persistent, antigen-specific, therapeutic antitumor immunity by dendritic cells genetically modified with an adenoviral vector to express a model tumor antigen.

Dendritic cells (DC) are potent antigen-presenting cells that play a critical role in the initiation of cellular immune responses. Using a BALB/c syngeneic colon carcinoma cell line expressing a model tumor antigen beta-galactosidase (betagal), we previously reported (Song et al, J Exp Med 1997; 186: 1247-1256) that immunization of mice with a single injection of DCs genetically modified with an adenovirus vector expressing betagal confers potent protection against a lethal intravenous tumor challenge, as well as suppression of pre-established lung tumors, resulting in a significant survival advantage. In the present study, we have addressed the question: how long does the memory of tumor antigen- specific immunity persists after DC priming in vivo using this genetically modified DC-based cancer vaccination strategy? To accomplish this, two groups of mice were evaluated: (1) mice surviving >400 days following protection from an initial intravenous tumor challenge after immunization with DC genetically modified to express betagal; and (2) mice surviving >300 days that had previously demonstrated regression of pre-established lung tumors after treatment with DC immunization. By analyzing the antigen-specific cytotoxic T lymphocyte response and challenging these long-term survival mice with a second subcutaneous tumor administration, the data demonstrate that a single administration of DC genetically modified to express a model antigen induces long-lasting, antigen-specific antitumor immunity in both naive and tumor-bearing hosts, observations that have important implications in the development of genetically modified DC-based antitumor vaccination strategies. Gene Therapy (2000) 7, 2080-2086.

Synergistic Induction of the Tap-1 Gene by IFN-γ and Lipopolysaccharide in Macrophages Is Regulated by STAT1

Proper regulation of the Tap-1 gene is critical for the initiation and continuation of a cellular immune response. Analysis of the Tap-1/low molecular mass polypeptide 2 bidirectional promoter showed that the IFN-gamma activation site element is critical for the rapid induction of the promoter by IFN-gamma following transfection into the human macrophage cell line THP-1. Furthermore, activation of STAT1 binding to this site was important for the synergistic response seen following the stimulation with both IFN-gamma and LPS. Mutation of an IFN-stimulated regulatory element that binds IFN regulatory factor 1 appeared to enhance the response to IFN-gamma and LPS. These data show that STAT1 is necessary for the activation of Tap-1 gene expression in APCs and initiation of cellular immune responses. Furthermore, our data suggest that bacterial products such as LPS may enhance cellular immune responses through augmenting the ability of STAT1 to regulate IFN-gamma-inducible genes.

Cellular immune response levels in gestational trophoblastic tumor patients after hydatidiform mole

Objective: This study looked for an association of natural (innate) cellular immune responses with the development of gestational trophoblastic tumors (GTT) after hydatidiform moles (HM). Method: An observational prospective cohort study (73 patients) was carried out to measure the serum level of Natural Killer (NK) cells, Monocytes (Mo) and a Bio-Merieux skin test was performed on the day of HM evacuation and 4 weeks afterwards. The diagnosis of GTT were after Soper and Hammond (1998) and followed up for at least 6 months after evacuation. Patients' characteristics and cellular immune responses among patients who developed GTT were analysed by multiple logistic regression (SPSS version 5). Results: The results showed that a low NK cell percentage was associated ( P=0.020) with the development of GTT, also a low Mo ( P=0.034) on day 0. While on day 28 a low NK cell percentage ( P=0.008) and Mo cell count ( P=0.043) were associated with the development of GTT. The cut-off point between the sensitivity (60%) and specificity of NK cell percentage on day 0 was 16, and also showed a negative predictive value of 83%. Meanwhile on day 28, the cut-off point between the sensitivity and specificity (80%) was 17, and showed a negative predictive value of NK cell percentage (85%). Conclusion: These figures indicated that HM-lowered NK cell serum percentages were associated with the development of GTT.

Activation and Suppression of Natural Cellular Immune Functions by Pneumocystis carinii

The regulatory role of soluble cytokines in innate cellular immune responses induced by Pneumocystis carinii was assessed in vitro in direct comparison to induction by Listeria monocytogenes. This report shows that P. carinii organisms, as well as L. monocytogenes, stimulated in whole spleen cell cultures of SCID mice the release of IFN-γ, TNF-α/β, IL-10, IL-12, and iNO. This response was independent of functional T cells. Both macrophages (MΦ) and natural killer (NK) cells were necessary for either microorganism to induce release of these cytokines. Co-cultures of purified MΦ - including alveolar MΦ - and purified NK cells indicated that no other cell population was necessarily involved. Microbial induction of NK cell-derived IFN-β has been reported to be mediated by the combined effects of TNF-α and IL-12 released by MΦ upon adequate microbial stimulation. Interestingly, only L. monocytogenes, but not P. carinii organisms could directly induce detectable amounts of TNF-α/β, IL-12, or iNO in purified MΦ cultures. In dose-response experiments, release of IFN-γ, TNF-α/β, and iNO was reduced at high relative concentrations of either microorganism. This high-dose suppression was at least partially controlled by MΦ-produced IL-10. Our data show that, P. carinii potently induces activating and inhibitory innate cellular immune response mechanisms and indicate that the initial step of macrophage-mediated NK cell activation might also involve other pathways than those described to date.

Immune Function and Cytochrome P4501A Activity after Acute Exposure to 3,3',4,4',5-Pentachlorobiphenyl (PCB 126) in Channel Catfish

Abstract Several commercial polychlorinated biphenyl (PCB) mixtures reduce immune functions in fish. However, little is known about the effects of specific coplanar PCBs on immune functions in fish. We investigated the effects of a single, acute exposure to either corn oil as a carrier control or 0.01, 0.1, and 1.0 mg/kg body weight of 3,3',4,4',5-pentachlorobiphenyl (PCB 126) on both nonspecific cellular immune responses and antigen-specific humoral immune responses in channel catfish Ictalurus punctatus. Fish were given PCB 126 intraperitoneally, and innate cellular immune responses were evaluated 3 and 7 d later. The humoral immune response against Edwardsiella ictaluri was evaluated 14 d after treatments. Immunological results were compared to cytochrome P4501A induction, ethoxyresorufin O-deethylase (EROD) activity, allometric indices, and hematology as biomarkers of exposure and effects 3 and 7 d posttreatment. Cytochrome P4501A and EROD activity were induced by all three doses of PCB 126, whereas a...

Specific T-cell factor production and lymphocytes in the direct surroundings of a subcutaneous allogeneic tumor

Antigen-specific T-cell factors (TCF) play a role in the initiation of cellular immune responses. In allogeneic mouse-tumor models lymphocytes from the direct tumor surroundings of both euthymic and nude mice produce TCF. These lymphocytes produce TCF when collected already 1 day after subcutaneous (sc) injection of tumor cells. In contrast to euthymic mice, draining lymph nodes and spleen of nude mice did not contain TCF-producing lymphocytes at any stage after sc tumor cell injection. In sensitized euthymic mice TCF production by lymphocytes is significantly higher in the direct tumor surroundings than in draining lymph nodes or spleen. At 2 and 5 days after tumor cell injection, the mononuclear cell infiltrate of the tissue surrounding the tumor in euthymic mice showed low expression of Thy 1, CD3, TCR αβ, TCR γδ, CD4, CD8, and asialo GM1, whereas several lymphocytes and mast cells were positive for monoclonal antibody (mAb) 14–30 (directed against TCF). In both euthymic and nude mice, sc injected tumor cells showed apoptosis. In conclusion, the direct tumor surroundings are the first (and, for nude mice, the only) site of TCF production, sc injection of tumor cells attracts mAb 14–30-positive lymphocytes and renders mast cells positive for mAb 14–30.