Interleukin-6 In Cultures Research Articles

Abstract A47: Superior expansion of central memory CD8+ T cells using NKG2D-targeted delivery of IL-2: Implications for adoptive T cell immunotherapy

Abstract Infusion of activated autologous lymphocytes or CAR-T cells is a promising clinical treatment for both solid and liquid tumors. Traditional methods for ex vivo T cell expansion have relied on transient stimulation of the T cell receptor in the presence of the common γ-chain cytokines. Although it has been routine for most clinical and experimental laboratories to rely on interleukin-2 (IL2) to support T cell expansion, this cytokine can result in expansion of regulatory T cells (Tregs) and activation induced death of effector cells. Furthermore, prolonged exposure to IL2 may result in differentiation of CD8+ T cells toward an effector phenotype, a terminally differentiated state that provides only short-lived and highly limited protection from malignancy. Alternate gamma chain cytokines, such as IL7 and IL15, do not expand Tregs and may offer an advantage for the expansion of central memory CD8+ T cells, that are superior in mediating tumor regression. Nevertheless, optimal protocols for T cell expansion have yet to be defined. We have recently demonstrated that IL2 targeted to NKG2D-expressing cells using the viral decoy ligand known as orthopox major histocompatibility complex class I-like protein (or OMCP for short), offers a superior method for NK cell expansion and NK mediated immunotherapy (Nat Commun 2016 Sep 21;7. doi: 10.1038/ncomms12878). The possibility of using this redirected cytokine (called OMCP-IL2 for short) for ex vivo CD8+ T cell expansion has not been explored. To directly compare several common gamma cytokines bulk T cells from C57BL/6 mice were cultured with transient CD3/CD28 stimulation in the presence of wild-type IL2, OMCP-IL2 or combination IL15/IL7 at 100IU/ml. While both IL2 and IL15/IL7 resulted in substantial T cell expansion by day 12 of culture (5.8±0.7 and 3.4±0.16-fold expansion respectively) OMCP-IL2 treated cultures resulted in 10±0.8 fold expansion by the same time point. Furthermore, IL2 and IL15/IL7-treated cultures did not expand further after two weeks of cytokine treatment while OMCP-IL2 treated cultures expanded for 40 days (reaching a 27±0.7-fold expansion). Flow cytometric analysis revealed that the higher CD8+ T cell numbers in OMCP-IL2 treated cultures were due to both increased proliferation (%KI67 positivity in 49±9%, 70±2% and 86±3% of CD8+ T cells for IL2, IL15/IL7 and OMCP-IL2 treated cultures respectively) and improved viability (27±2%, 20±2% and 79±1.8% viable cells for IL2, IL15/IL7 and OMCP-IL2-treated cultures respectively). While wild-type IL2 expanded both CD8+ and CD4+ T cells OMCP-IL2 preferentially expanded CD8+ T cells. Phenotypic analysis revealed that the majority of CD8+ T cells expanded in OMCP-IL2 were CD44hiCD62Lhi central memory T cells while those expanded in IL2 were primarily CD44hiCD62Llow effector cells. Consistent with this a starting population of 1 million T cells resulted in 60,756±21,813; 368,756±30,217; and 7,605,870±872,870 CD8+ central memory T cells after three weeks of culture in IL2, IL15/7 and OMCP-IL2 respectively. Furthermore, both IL2 and IL15/7 expanded cells expressed high levels of surface PD-1 (67.8% and 40.9% respectively) while minimum PD-1 was expressed on OMCP-IL2 expanded cells (18.4%) In summary, we now demonstrate that the use of an NKG2D-targeted form of IL2 provides a significant quantitative and qualitative advantage for CD8+ T cell expansion in vitro. Such an advantage might be the result of precise delivery of IL2 to cytotoxic T cells or novel signaling pathways induced by engagement of both the NKG2D and IL2 receptor. Since the ability to obtain a significant number of CAR T cells limits the clinical application of this technology the possibility of quickly and efficiently expanding T cells will be key to improving clinical therapy. Citation Format: Kang Li, Lei Shi, Qing Wang, Oscar Onyema, Yizhan Guo, Alexander Sasha Krupnick. Superior expansion of central memory CD8+ T cells using NKG2D-targeted delivery of IL-2: Implications for adoptive T cell immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A47.

Modulation of intracellular expression of IFNγ and IL-2 in culture of splenic T lymphocytes by some flavonoid glycosides of Alchornea floribunda

Context Alchornea floribunda Müll. Arg. (Euphorbiaceae) leaves are widely used in ethnomedicine for the management of rheumatism, arthritis and toothache.Objective In this study, flavonoid glycosides isolated from Alchornea floribunda were screened for their effect on the intracellular expression of interferon-gamma (IFNγ) and interleukin-2 (IL-2) type-1 cytokines.Materials and methods Chromatographic purification of the ethyl acetate fraction of the methanol leaf extract led to the isolation of seven flavonoid glycosides (1–7). Their structures were elucidated by 1D and 2D nuclear magnetic resonance and mass spectrometry. Splenocytes were treated with graded concentrations of the compounds (6.25–25 μg/mL) and incubated for 24 h. Thereafter, their effect on the expression of IFNγ and IL-2 by CD4+ and CD8+ T-lymphocytes was evaluated using intracellular cytokine staining and FACS analysis.Results Compounds 1–7 (6.25–25 μg/mL) caused the up-regulation of activated CD8+ (57.85–72.45% versus 57.85% for untreated control) and, to a lesser extent, activated CD4+ (3.21–7.21% versus 2.75% for the untreated control) T-lymphocytes that were both largely interferon-gamma-releasing in treated mouse T lymphocytes relative to untreated control. FACS data analysis showed that stimulation with all the compounds increased the proportion of CD8+/IFNγ+ and CD4+/IFNγ+ T lymphocytes up to two-fold when compared with the cells in untreated control wells. Intracellular IL-2 secretion by treated T cells was not detected.Conclusion This recorded T-lymphocyte-specific immune-modulatory property may contribute to explain in part the dynamics associated with the ethnomedicine of Alchornea floribunda, and may find relevance as a necessary cellular immune response precursor to infection-associated disease management.

Selection of CD8+PD-1+ Lymphocytes in Fresh Human Melanomas Enriches for Tumor-reactive T Cells

CD8+ tumor-infiltrating lymphocytes (TILs) in human melanomas express high levels of PD-1 and are functionally impaired. However, adoptive cell therapy using in vitro-expanded TIL can be a highly effective therapy for patients with advanced melanoma. This discrepancy led us to further analyze the CD8+PD-1+ TILs. We found that the percentage of PD-1-expressing CD8+ T cells was higher in the tumor digests that generate tumor-reactive TILs after in vitro culture in interleukin-2 (P=0.0007). Also sorted and expanded CD8+PD-1+ T cells in tumor digests showed much higher tumor-specific interferon-γ production compared with CD8+PD-1⁻ T cells. These results suggested that tumor-specific CD8+ T cells in melanoma tumor digests are largely PD-1, and this population can recover function after culturing in interleukin-2. PD-1 has been reported as an inhibitory receptor on T cells. We found that the in vitro functional suppression of cultured-TILs from native levels of PD-L1 expression on melanomas was minimal, and moreover expression level of PD-1 on CD8+ tumor-specific TILs decreased during the culture. As a consequence, the PD-1 receptor can be a useful biomarker for enriching tumor-specific T cells from fresh melanomas.

Establishment and Characterization of an Experimental Model of Coronary Thrombotic Microembolism in Rats

To establish a model of coronary thrombotic microembolism in rats, either automicrothrombotic particulates (CM group) or saline control (SHAM group) was injected into temporarily clamped aortas of male Sprague-Dawley rats. After automicrothrombotic particulate injection, serum c-troponin I and von Willebrand factor levels, the no-flow area as evaluated by Thioflavin S, myocardial leukocyte infiltration levels, myocardial expressions of tumor necrosis factor alpha and interleukin-6, the percentage of arterioles obstructed by thrombosis, and myocardial fibrosis were all significantly increased whereas cardiac function as evaluated by echocardiography and hemodynamic measurements were significantly reduced compared with the sham group. Thus, aortic automicrothrombotic particulate injection could induce coronary microembolism in rats, and this model could be of value in improving the understanding of pathophysiology of coronary microembolism.

Clinical Expansion of Cord Blood-derived T Cells for Use as Donor Lymphocyte Infusion After Cord Blood Transplantation

Allogeneic stem cell transplantation (SCT) from cord blood (CB) as a stem cell source is a promising alternative when no human leukocyte antigen-matched donor is found. Donor lymphocyte infusion (DLI) is a possible treatment modality for threatening graft failure or relapse of an underlying malignancy after transplantation. Ethical and logistical reasons limit the possibility of DLI in the setting of CB SCT. To remedy this restriction, we performed expansion of donor T cells in vitro from CB grafts in a clinical setting for use as future DLI and characterized the expanded cells in comparison to T cells from CB acquired ex vivo and adult peripheral blood. T cells were expanded from grafts used for transplantation, upon CD3/CD28 crosslinking and culture in interleukin-2. Phenotype and function of T cells were assessed by flow cytometry and mixed lymphocyte culture assays. T-cell receptor repertoire distribution was evaluated with polymerase chain reaction-based spectratyping. We were able to amplify T cells to sufficient amounts for DLI in 13 out of 13 initiated expansions. Expanded T cells presented with an activated phenotype and could be induced to produce cytokines by a nonspecific stimulus. When exposed to allogeneic targets, expanded CB T cells proliferated at comparable levels to their ex vivo and adult blood counterparts. In summary, clinical expansion of CB T cells for DLI is feasible and may be a future modality for treatment of graft failure or relapse after SCT.

The immunosuppressive activities of newly synthesized azaphenothiazines in human and mouse models

In this study, we evaluated the activities of new types of azaphenothiazines in the following immunological assays: the proliferative response of human peripheral blood mononuclear cells induced by phytohemagglutin A or anti-CD3 antibodies; lipopolysaccharide-induced cytokine production by human PBMC; the secondary, humoral immune response in mice to sheep erythrocytes (in vitro); and delayed-type hypersensitivity in mice to ovalbumin (in vivo). In some tests, chlorpromazine served as a reference drug. The compounds exhibited differential inhibitory activities in the proliferation tests, with 10H-2,7-diazaphenothiazine (compound 1) and 6-(3-dimethylaminopropyl)diquinothiazine (compound 8) being most suppressive. Compound 1 was selected for further studies, and was found to be strongly suppressive in the humoral immune response even at low concentrations (1 μg/ml). Compound 1 also inhibited the delayed-type hypersensitivity lipopolysaccharide-induced production of tumor necrosis factor and interleukin-6 in cultures of human blood cells. As there were only two subjects in this study, the effects of these compounds on human blood cells need to be confirmed. In this paper, we also discuss the structure-activity relationships of selected compounds.

Enhancement of in vitrointerleukin-2 production in normal subjects following a single spinal manipulative treatment

BackgroundIncreasing evidence supports somato-visceral effects of manual therapies. We have previously demonstrated that a single spinal manipulative treatment (SMT) accompanied by audible release has an inhibitory effect on the production of proinflammatory cytokines in asymptomatic subjects. The purpose of this study is to report on SMT-related changes in the production of the immunoregulatory cytokine interleukin 2 (IL-2) and to investigate whether such changes might differ with respect to the treatment approach related to the presence or absence of an audible release (joint cavitation).MethodsOf 76 asymptomatic subjects, 29 received SMT with cavitation (SMT-C), 23 were treated with SMT without cavitation (SMT-NC) and 24 comprised the venipuncture control (VC) group. The SMT-C and SMT-NC subjects received a single, similar force high velocity low amplitude manipulation, in the upper thoracic spine. However, in SMT-NC subjects, positioning and line of drive were not conducive to cavitation. Blood and serum samples were obtained before and then at 20 and 120 min post-intervention. The production of IL-2 in peripheral blood mononuclear cell cultures was induced by activation for 48 hr with Staphylococcal protein A (SPA) and, in parallel preparations, with the combination of phorbol ester (TPA) and calcium ionophore. The levels of IL-2 in culture supernatants and serum were assessed by specific immunoassays.ResultsCompared with VC and their respective baselines, SPA-induced secretion of IL-2 increased significantly in cultures established from both SMT-C and SMT-NC subjects at 20 min post-intervention. At 2 hr post-treatment, significant elevation of IL-2 synthesis was still apparent in preparations from SMT-treated groups though it became somewhat attenuated in SMT-NC subjects. Conversely, IL-2 synthesis induced by TPA and calcium ionophore was unaltered by either type of SMT and was comparable to that in VC group at all time points. No significant alterations in serum-associated IL-2 levels were observed in any of the study groups.ConclusionThe present study demonstrates that, the in vitro T lymphocyte response to a conventional mitogen (SPA), as measured by IL-2 synthesis, can become enhanced following SMT. Furthermore, within a period of time following the manipulative intervention, this effect may be independent of joint cavitation. Thus the results of this study suggest that, under certain physiological conditions, SMT might influence IL-2-regulated biological responses.

Activation of c-Kit in dendritic cells regulates T helper cell differentiation and allergic asthma

Dendritic cells (DCs) are integral to the differentiation of T helper cells into T helper type 1 T(H)1, T(H)2 and T(H)17 subsets. Interleukin-6 (IL-6) plays an important part in regulating these three arms of the immune response by limiting the T(H)1 response and promoting the T(H)2 and T(H)17 responses. In this study, we investigated pathways in DCs that promote IL-6 production. We show that the allergen house dust mite (HDM) or the mucosal adjuvant cholera toxin promotes cell surface expression of c-Kit and its ligand, stem cell factor (SCF), on DCs. This dual upregulation of c-Kit and SCF results in sustained signaling downstream of c-Kit, promoting IL-6 secretion. Intranasal administration of antigen into c-Kit-mutant mice or neutralization of IL-6 in cultures established from the lung-draining lymph nodes of immunized wild-type mice blunted the T(H)2 and T(H)17 responses. DCs lacking functional c-Kit or those unable to express membrane-bound SCF secreted lower amounts of IL-6 in response to HDM or cholera toxin. DCs expressing nonfunctional c-Kit were unable to induce a robust T(H)2 or T(H)17 response and elicited diminished allergic airway inflammation when adoptively transferred into mice. Expression of the Notch ligand Jagged-2, which has been associated with T(H)2 differentiation, was blunted in DCs from c-Kit-mutant mice. c-Kit upregulation was specifically induced by T(H)2- and T(H)17-skewing stimuli, as the T(H)1-inducing adjuvant, CpG oligodeoxynucleotide, did not promote either c-Kit or Jagged-2 expression. DCs generated from mice expressing a catalytically inactive form of the p110delta subunit of phosphatidylinositol-3 (PI3) kinase (p110(D910A)) secreted lower amounts of IL-6 upon stimulation with cholera toxin. Collectively, these results highlight the importance of the c-Kit-PI3 kinase-IL-6 signaling axis in DCs in regulating T cell responses.

Reduced Natural Killer Function Accompanies MDS Disease Progression and Is Restored by Lenalidomide (CC5013, Revlimid®) Via a Mechanism Divergent from Interleukin-2 (IL-2).

Background: Patients with myelodysplastic syndromes (MDS) display several abnormalities of T cell and NK cellular immunity. Tumor lysis by NK cells occurs through orchestrated control by inhibitory NK receptors (NKRs) and activating NKRs. Lenalidomide is an immunomodulatory (IMiD) drug structurally related to thalidomide, which has proven clinical efficacy for the treatment of low-risk MDS (List et al, NEJM 2005; 352:549). Investigations in patients with multiple myeloma (MM) reported that thalidomide was associated with enhanced cytolytic function and increased NK cells in responding patients. We investigated the action of lenalidomide on NK function and activating NK receptor expression in patients with MDS.Methods: MDS patients were categorized according to WHO category, age, sex, IPSS, and cytogenetics. Peripheral blood cells (PBMCs) were isolated from patients and normal donors and NK receptor expression determined in paired healthy and patient PBMCs by 3-color flow cytometry. NK receptor expression was analyzed for CD158a (KIR2DL1, KIR2DS1), CD158b (KIR2DL2, KIR2DL3, KIR2DL3), KIR3DL1, KIR2DS4, NKG2A, NKG2D, NKp30, NKp44, and NKp46. Cytotoxicity assays were performed using 5-hour 51Cr-release assays.Results: Forty-eight MDS patients and 37 normal donors were analyzed, demonstrating that NK cytolytic function was reduced in the patient population (19% ± 21 S.D. vs. 44% ± 21) (p<0.001). Reduced NK function in MDS was significantly associated with higher IPSS (p=0.01), abnormal karyotype (p=0.05), presence of excess blasts (p=0.01), and age-adjusted bone marrow hypercellularity (p=0.04). MDS patients had reduced display of the activating receptor NKp30, accompanied by NKG2D (an activating C lectin-like (NKG2)-family receptor) downregulation that closely correlated with impaired NK function (p=0.001). NK-mediated cytotoxicity and NKR expression were examined after treatment with lenalidomide 5μM for 72 hours or IL-2 in normal donors and MDS patients. Lenalidomide augmented NK lytic function in normal donors (vehicle, 42% ± 15 vs. lenalidomide, 71% ± 17; P≤0.01 by t test) and in 10 out of 17 (59%) of the MDS patients (vehicle, 25% ± 25 vs. lenalidomide 42% ± 33). IL-2 increased NK cytolytic function in all MDS patients (vehicle, 16% ± 16 vs. lenalidomide, 47% ± 33) and the percentage of NK cells expressing CD69 was increased from both MDS patients and normal controls. Similarly, the percentage of NK cells expressing NKp30, NKG2D, NKp44 but not NKp46 was significantly enhanced by IL-2 culture. In contrast, lenalidomide treatment failed to induce NKR expression in MDS patients and normal controls.Conclusions: These findings suggest that impairment of NK cytolytic function in MDS derives in part from reduced display of the activating NK receptors such as NKG2D, which accompanies disease progression. Lenalidomide augments NK function in some patients; however, the mechanisms of functional augmentation by IL-2 and lenalidomide are divergent. Evasion of NK immunosurveillance may be an important factor associated with MDS disease progression.

Kupffer Cells Abrogate Cholestatic Liver Injury in Mice

Biliary obstruction and cholestasis can cause hepatocellular apoptosis and necrosis. Ligation of the common bile duct in mice provides an excellent model in which to study the underlying mechanisms. Kupffer cells play a key role in modulating the inflammatory response observed in most animal models of liver injury. This study was performed to determine the role of Kupffer cells in the injury attending cholestasis. Mice were not treated or were rendered Kupffer cell-depleted by intravenous inoculation of multilamellar liposome-encapsulated dichloromethylene diphosphonate, the common bile duct was ligated and divided; sham-operated animals served as controls. Similarly, interleukin-6 (IL-6)-deficient and tumor necrosis factor-receptor-deficient mice underwent bile duct ligation (BDL) or sham operations. Serum alanine transaminase levels were increased in all BDL mice at 3 days after surgery, but were significantly higher in IL-6-deficient mice or mice rendered Kupffer cell-depleted before ligation. Histologic examination of BDL livers showed portal inflammation, neutrophil infiltration, bile duct proliferation, and hepatocellular necrosis. Photoimage analyses confirmed more necrosis in the livers of Kupffer cell-depleted and IL-6-deficient animals. Purified Kupffer cells derived from BDL animals produced more IL-6 in culture. Similarly, Kupffer cells obtained by laser capture microdissection from the livers of BDL mice expressed increased levels of IL-6 messenger RNA. Recombinant mouse IL-6 administered 1 hour before BDL completely reversed the increased liver damage assessed otherwise in Kupffer cell-depleted mice. These findings indicate that Kupffer cells abrogate cholestatic liver injury by cytokine-dependent mechanisms that include the production of IL-6.

Expression and function of KIR and natural cytotoxicity receptors in NK-type lymphoproliferative diseases of granular lymphocytes

AbstractUsing monoclonal antibodies (mAbs) specific for different natural killer (NK) receptors, we studied the lymphocyte population from 18 patients with NK-type lymphoproliferative disease of granular lymphocytes (LDGL). The analysis of both resting and cultured NK cell populations demonstrated that these patients are frequently characterized by NK cells displaying a homogeneous staining with given anti–killer Ig-like receptor (anti-KIR) mAb (11 of 18 patients). In most patients NK cells were characterized by the CD94/NKG2A+ phenotype, whereas only a minor fraction of the cases expressed CD94/NKG2C. In 7 of these patients we could also assess the function of the various NK receptors. Remarkably those KIR molecules that, in each patient, homogeneously marked the NK cell expansion were found to display an activating function as determined by cross-linking with specific anti-KIR mAb. The KIR genotype analysis performed in 13 of 18 cases revealed that in NK-type LDGL certain activating KIRs, as well as certain infrequent KIR genotypes, were detected with higher frequencies as compared to previously analyzed healthy donors. Moreover, most KIR genotypes included multiple genes coding for activating KIRs. The analysis of non–HLA-specific triggering receptors indicated that the natural cytotoxicity receptors (NKp46, NKp30) were expressed at significantly low levels in freshly drawn NK cells from most patients analyzed. However, in most instances the expression of NKp46 and NKp30 could be up-regulated on culture in interleukin 2. Our data indicate that in NK-LDGL the expanded subset is frequently characterized by the expression of a given activating KIR, suggesting a direct role for these molecules in the pathogenetic mechanisms of this disorder.

Restored T-cell activation mechanisms in human tumour-infiltrating lymphocytes from melanomas and colorectal carcinomas after exposure to interleukin-2.

We investigated the effects of interleukin-2 (IL-2) exposure on T-cell signal transduction molecules and apoptosis markers in tumour-infiltrating lymphocytes (TIL) isolated from 20 melanoma and 16 colorectal carcinoma metastases and expanded in vitro for therapeutic reinfusion. Before IL-2 culture, TIL showed undetectable or very low levels of T-cell receptor (TCR) ɛ chain, p56lck, Fas ligand (FasL) and Bax expression, while Bcl-2 values were elevated. Cancer cells were characterised by low or absent Fas and Bcl-2 and high Bax expression. Notably, they also expressed FasL. After 41–48 days of IL-2 culture, TCR ɛ chain and p56lck expression of TIL rose to median values of approximately 80 and 30% positive cells, respectively (P<0.001), FasL expression was detected in 45% cells from melanomas (P<0.001) and in 3% from colorectal carcinomas (P=0.09), and Bax-positive cells increased from 17.5 to 70% (P=0.005). Moreover, TCR ζ chain-positive cells were significantly increased from baseline (P=0.001), Bcl-2-positive cells dropped from 50 to 1% (P=0.007) and perforin content was high, while Fas expression was not significantly modified by IL-2 culture. In conclusion, our data suggest that the degree of immunosuppression in TIL from melanomas and colorectal carcinomas is very high, and the apoptosis markers' repertoire of cancer cells resembles that of immune-privileged tissue. Interleukin-2 culture appears to restore lymphocyte activation mechanisms, resulting in consistent FasL expression and perforin production.

Depletion of CD4+ CD25+ regulatory cells augments the generation of specific immune T cells in tumor-draining lymph nodes.

Recent studies have identified a unique population of CD4+CD25+ regulatory T cells that is crucial for the prevention of spontaneous autoimmune diseases. Further studies demonstrated that depletion of CD4+CD25+ T cells enhances immune responses to nonself antigens. Because immune responses to malignant tumors are weak and ineffective, depletion of regulatory T cells has been reported to result in tumor regression. In the current study, using the weakly immunogenic MCA205 sarcoma and the poorly immunogenic B16/BL6/D5 (D5) melanoma, depletion of CD4+CD25+ T cells by the administration of anti-CD25 monoclonal antibodies (mAb), PC61 induced some tumor growth retardation, but all mice eventually succumbed to tumors. In our laboratory, immunotherapy by the transfer of tumor-immune T cells has demonstrated potent antitumor effects. A reliable source of tumor-reactive T cells has been lymph nodes (LN) draining progressive tumors. Therapeutic effector T cells can be generated by in vitro activation of draining LN cells with anti-CD3 mAb followed by culture in interleukin-2. In this system, PC61 mAb depletion of CD4+CD25+ T cells before or on day 8 of tumor growth resulted in increased sensitization in the draining LN. The therapeutic efficacy of activated tumor-draining LN cells from mAb depleted mice increased approximately three fold while maintaining specificity when tested in adoptive immunotherapy of established pulmonary metastases. Specific interferon-gamma secretion by LN T cells from mice treated with PC61 mAb 1 day before tumor inoculation increased significantly. However, this increase was not demonstrated with LN T cells from mice treated on day 8 despite their enhanced therapeutic reactivities. Our results indicate that although the antitumor immunity enhanced by the depletion of CD4+CD25+ T cells is insufficient to eradicate tumors, it augments the sensitization of immune T cells in the draining LN, thus, facilitating adoptive immunotherapy.

The plasmid pcDNA3 differentially induces production of interferon-α and interleukin-6 in cultures of porcine leukocytes

An adjuvant effect of invertebrate DNA has been attributed to its relative high frequency of unmethylated CpG dinucleotides. Here we describe the interferon-α (IFN-α) and interleukin-6 (IL-6) inducing properties of a commonly used eukaryotic expression vector, pcDNA3, in porcine leukocytes. The magnitude of the cytokine response was compared to that induced by the synthetic ds RNA analogue poly(I):poly(C), inactivated preparations of Aujeszky’s disease virus (ADV) and the Gram-negative bacteria Actinobacillus pleuropneumoniae. The plasmid, as well as poly(I):poly(C), required lipofectin to induce IFN-α production whereas both preparations induced IL-6 irrespective of preincubation with lipofectin. However, the nucleic acid-induced levels of IL-6 were low compared to those induced by A. pleuropneumoniae. The IFN-α response elicited by pcDNA3 in the presence of lipofectin was as high as, or higher than that induced by ADV. Interestingly, also A. pleuropneumoniae induced a substantial production of IFN-α when preincubated with lipofectin. Plasmid expression was not necessary for induction of IFN-α. Furthermore, the IFN-α inducing capacity of pcDNA3 was not reduced when the two predicted immunostimulatory sequences 5′AACGTT3′ were deleted. Nor did the ability of the plasmid to induce IFN-α production decrease when the ampicillin resistance (ampR) gene was replaced with the kanamycin resistance (kanR) gene. However, methylation of all cytidines in CpG dinucleotides of pcDNA3 abolished the IFN-α inducing capacity. These in vitro results indicate an immunomodulatory role of bacterial DNA also in the pig. Unmethylated CpG dinucleotides are crucial for induction of IFN-α by the plasmid, but other CpG motifs than those within the 5′AACGTT3′ sequences of the ampR gene contribute to this induction in porcine cells.

Delivery of liposome-encapsulated HIV type 1 proteins to human dendritic cells for stimulation of HIV type 1-specific memory cytotoxic T lymphocyte responses.

An important aspect of vaccine development involves delivery of antigens to antigen-presenting cells for the induction of potent antigen-specific T lymphocyte responses. We investigated the effect of a cationic liposome, lipofectin, on delivery of whole proteins to human dendritic cells (DCs) derived from blood mononuclear cells by culture in interleukin 4 and granulocyte-monocyte colony-stimulating factor for stimulation of human immunodeficiency virus type 1 (HIV-1)-specific memory cytotoxic T lymphocyte (CTL) responses. Delivery of HIV-1 Gag, Pol, and Env proteins to DCs by lipofectin stimulated greater anti-HIV-1 memory CTL responses in cells from HIV-1-infected subjects than those induced by DCs loaded with protein alone. The CTLs were CD8+ and HLA class I restricted. Antigen presentation was enhanced by chloroquine, but blocked by brefeldin A and peptide aldehyde inhibitors of proteasomes, indicating that the classic MHC class I cytosolic pathway was used for processing and presentation of HIV-1 protein by the DCs. Stimulation of anti-HIV-1 CTLs by this safe, inexpensive, and broadly applicable approach may be used in DC-based therapies for HIV-1 infection.

Cardiac myocytes produce interleukin-6 in culture and in viable border zone of reperfused infarctions.

Previous work from our laboratory demonstrated that interleukin (IL)-6 plays a potentially critical role in postreperfusion myocardial injury and is the major cytokine responsible for induction of intracellular adhesion molecule (ICAM)-1 on cardiac myocytes during reperfusion. Myocyte ICAM-1 induction is necessary for neutrophil-associated myocyte injury. We have previously demonstrated the induction of IL-6 in the ischemic myocardium, and the current study addresses the cells of origin of IL-6. In the present study, we combined Northern blot analysis and in situ hybridization to demonstrate IL-6 gene expression in cardiac myocytes. Isolated ventricular myocytes were stimulated with tumor necrosis factor-alpha, IL-1beta, lipopolysaccharide, preischemic lymph, and postischemic lymph. Unstimulated myocytes showed no significant IL-6 mRNA expression. Myocytes stimulated with preischemic lymph showed minimal or no IL-6 mRNA expression, whereas myocytes stimulated with tumor necrosis factor-alpha, IL-1beta, lipopolysaccharide, or postischemic lymph showed a strong IL-6 mRNA induction. Northern blot with ICAM-1 probe revealed ICAM-1 expression under every condition that demonstrated IL-6 induction. We then investigated the expression of IL-6 mRNA in our canine model of ischemia and reperfusion. Cardiac myocytes in the viable border zone of a myocardial infarction exhibited reperfusion-dependent expression of IL-6 mRNA within 1 hour after reperfusion. Mononuclear cells infiltrate the border zone and express IL-6 mRNA. Isolated cardiac myocytes produce IL-6 mRNA in response to several cytokines as well as postischemic cardiac lymph. In addition to its production by inflammatory cells, we demonstrate that IL-6 mRNA is induced in myocytes in the viable border zone of a myocardial infarct. The potential roles of IL-6 in cardiac myocytes in an infarct border are discussed.

Production of Interleukin-6 in Cultures of Peripheral Blood Mononuclear Cells from Children with Primary Protein-Calorie Malnutrition and from Eutrophic Controls

The spontaneous as well as mitogen-induced in vitro production of interleukin-6 (IL-6) was studied in cultures of peripheral blood mononuclear cells (PBMC) from 14 children with marginal protein-energy malnutrition, 43 children with definite protein-energy malnutrition and 38 eutrophic controls of similar age, sex, race and socioeconomical condition. PBMC were cultured without added mitogen or stimulated with either lipopolysaccharide (LPS) or phytohemagglutinin (PHA). After 48 h incubation, cell-free culture supernatants were collected and stored at –70°C. The amount of IL-6 in the supernatants was determined by a specific bioassay based on the proliferation of B9 hybridoma cells using human rIL-6 as standard. The mean level of IL-6 was significantly increased in supernatants from nonstimulated PBMC cultures from definitely malnourished children as compared with that observed in those of the controls. Stimulation with either LPS or PHA induced a rise in cytokine bioactivity in the supernatants of PBMC cultures from the different nutritional groups tested. Interestingly, IL-6 was significantly increased in the supernatants of PHA-stimulated cultures from malnourished children as compared with those of the controls.

NKp44, a novel triggering surface molecule specifically expressed by activated natural killer cells, is involved in non-major histocompatibility complex-restricted tumor cell lysis.

After culture in interleukin (IL)-2, natural killer (NK) cells acquire an increased capability of mediating non-major histocompatibility complex (MHC)-restricted tumor cell lysis. This may reflect, at least in part, the de novo expression by NK cells of triggering receptors involved in cytolysis. In this study we identified a novel 44-kD surface molecule (NKp44) that is absent in freshly isolated peripheral blood lymphocytes but is progressively expressed by all NK cells in vitro after culture in IL-2. Different from other markers of cell activation such as CD69 or VLA.2, NKp44 is absent in activated T lymphocytes or T cell clones. Since NKp44 was not detected in any of the other cell lineages analyzed, it appears as the first marker specific for activated human NK cells. Monoclonal antibody (mAb)-mediated cross-linking of NKp44 in cloned NK cells resulted in strong activation of target cell lysis in a redirected killing assay. This data indicated that NKp44 can mediate triggering of NK cell cytotoxicity. mAb-mediated masking of NKp44 resulted in partial inhibition of cytolytic activity against certain (FcgammaR-negative) NK-susceptible target cells. This inhibition was greatly increased by the simultaneous masking of p46, another recently identified NK-specific triggering surface molecule. These data strongly suggest that NKp44 functions as a triggering receptor selectively expressed by activated NK cells that, together with p46, may be involved in the process of non-MHC-restricted lysis. Finally, we show that p46 and NKp44 are coupled to the intracytoplasmic transduction machinery via the association with CD3zeta or KARAP/DAP12, respectively; these associated molecules are tyrosine phosphorylated upon NK cell stimulation.

Differential Cytotoxicity of Cord Blood and Bone Marrow–Derived Natural Killer Cells

Allogeneic cord blood is now being widely used as a source of stem cells for hematologic reconstitution after myeloablative therapy, with reported significantly lower levels of graft-versus-host disease (GVHD) compared with the use of allogeneic bone marrow (BM). This study was undertaken to investigate biologic aspects of natural killer (NK) cell activity, as recognized effector cells of the GVHD and graft-versus-leukemia (GVL) response, from cord blood and conventional BM. NK-cell activity levels of freshly isolated cells from cord blood and BM against K562 targets were comparable. Lymphokine activated killer (LAK) cells from both hematopoietic cell sources were compared for their ability to kill target cells by necrotic or apoptotic mechanisms using specific target cell lines. Cord blood cells had significantly higher necrosis-mediated cytotoxic activity against Daudi target cells compared with BM-derived cells. Cord blood LAK cells had relatively high levels of apoptotic-mediated cytotoxicity against YAC-1 target cells, whereas BM-derived LAK cells were unable to induce apoptosis in these cells. Interleukin-2 (IL-2) induced significant granzyme B activity in cord cells in contrast to BM cells, in which very little activity was measured. Western blotting confirmed these findings, with IL-2 inducing granzyme B protein expression in cord cells but not detectable levels in BM cells. BM cells had significantly lower cell surface expression of IL-2R and prolonged culture in IL-2 was only partially able to restore their deficient apoptotic cytotoxic activity. Thus, major differences exist between cord blood-derived and BM-derived mononuclear cells with respect to their NK-cell-associated cytotoxic behavior. This could have important implications for stem cell transplantation phenomena, because it suggests that cord blood may have increased potential for a GVL effect.

Differential Cytotoxicity of Cord Blood and Bone Marrow–Derived Natural Killer Cells

AbstractAllogeneic cord blood is now being widely used as a source of stem cells for hematologic reconstitution after myeloablative therapy, with reported significantly lower levels of graft-versus-host disease (GVHD) compared with the use of allogeneic bone marrow (BM). This study was undertaken to investigate biologic aspects of natural killer (NK) cell activity, as recognized effector cells of the GVHD and graft-versus-leukemia (GVL) response, from cord blood and conventional BM. NK-cell activity levels of freshly isolated cells from cord blood and BM against K562 targets were comparable. Lymphokine activated killer (LAK) cells from both hematopoietic cell sources were compared for their ability to kill target cells by necrotic or apoptotic mechanisms using specific target cell lines. Cord blood cells had significantly higher necrosis-mediated cytotoxic activity against Daudi target cells compared with BM-derived cells. Cord blood LAK cells had relatively high levels of apoptotic-mediated cytotoxicity against YAC-1 target cells, whereas BM-derived LAK cells were unable to induce apoptosis in these cells. Interleukin-2 (IL-2) induced significant granzyme B activity in cord cells in contrast to BM cells, in which very little activity was measured. Western blotting confirmed these findings, with IL-2 inducing granzyme B protein expression in cord cells but not detectable levels in BM cells. BM cells had significantly lower cell surface expression of IL-2R and prolonged culture in IL-2 was only partially able to restore their deficient apoptotic cytotoxic activity. Thus, major differences exist between cord blood-derived and BM-derived mononuclear cells with respect to their NK-cell–associated cytotoxic behavior. This could have important implications for stem cell transplantation phenomena, because it suggests that cord blood may have increased potential for a GVL effect.