Addition Of Interleukin-2 Research Articles

Postnatal Acquisition of Primary Rhesus Cytomegalovirus Infection is Associated With Prolonged Virus Shedding and Impaired CD4+ T Lymphocyte Function

Although virus-specific CD4(+) T lymphocytes emerge rapidly during primary cytomegalovirus (CMV) infection in humans, they exhibit a state of prolonged functional exhaustion of unknown etiology. To investigate the suitability of rhesus macaques as a model of primary human CMV infection, we examined the virologic and immunologic features of naturally acquired primary CMV infection in rhesus macaques. CMV-specific CD4(+) T lymphocytes and CMV load in blood, saliva, and urine were evaluated in a cohort of simian immunodeficiency virus (SIV)-negative rhesus macaques stratified by age into infant, juvenile, and adult groups. CMV infection was detected in juvenile and adult monkeys but not in infant monkeys. CMV loads and shedding frequency in urine and saliva were significantly higher in the 2-3-year old juvenile monkeys, compared with the adult monkeys. The increased CMV load in juvenile monkeys was associated with lower polyfunctionality, impaired proliferation, and increased expression of the inhibitory receptor PD-1 in CMV-specific CD4(+) T lymphocytes. The proliferative defect was partially reversible by exogenous PD-1 blockade or addition of interleukin 2. Postnatal acquisition of primary CMV infection in rhesus macaques results in prolonged virus excretion and impaired CMV-specific CD4(+) T-lymphocyte function, findings that recapitulate key features of primary CMV infection in humans.

Zoledronate Sensitizes Neuroblastoma-derived Tumor-initiating Cells to Cytolysis Mediated by Human γδ T Cells

Neuroblastoma is the most common extracranial solid tumor in children that is refractory to intensive multimodal therapy. In particular, tumor-initiating cells (TICs) derived from neuroblastoma are believed responsible for tumor formation and resistance to the conventional therapy; an optimal strategy therefore should target this population. Technically, TICs can be enriched from neuroblastoma-derived spheres when the tumor cells are cultured in a serum-free medium supplemented with certain growth factors. Recently, a line of evidence has suggested antitumor potential of Vγ9Vδ2 T cells (γδ T cells), a T-cell population that recognizes and kills target cells independent of surface HLA expressions. Furthermore, a mevalonate pathway inhibitor, zoledronate, has been reported to enhance cytolytic activity of γδ T cells. On the basis of these findings, we hypothesized that zoledronate would sensitize neuroblastoma TICs to γδ T-cell-mediated cytolysis and promote therapeutic efficacy against neuroblastoma. In the current study, we show that zoledronate efficiently sensitizes both neuroblastoma-derived adherent cells and sphere-forming cells to γδ T-cell-mediated cytolysis. Subsequently, in vitro colony formation inhibition assay and in vivo animal studies reveal that the presence of γδ T cells decelerates outgrowth of neuroblastoma TICs. We finally show that addition of interleukin-15 and/or interleukin-18 in culture enhances the cytolytic activity of γδ T cells. On the basis of these data, we conclude that ex vivo expanded γδ T cells are a promising tool for antineuroblastoma immunotherapy with options for further improvement.

Revision joint replacement, wear particles, and macrophage polarization

Currently, younger, more active patients are being offered total joint replacement (TJR) for end-stage arthritic disorders. Despite improved durability of TJRs, particle-associated wear of the bearing surfaces continues to be associated with particulate debris, which can activate monocyte/macrophages. Activated macrophages then produce pro-inflammatory factors and cytokines that induce an inflammatory reaction that activates osteoclasts leading to bone breakdown and aseptic loosening. We hypothesized that activated macrophages in tissues harvested from revised joint replacements predominantly express an M1 pro-inflammatory phenotype due to wear-particle-associated cell activation, rather than an M2 anti-inflammatory phenotype. We further questioned whether it is possible to convert uncommitted monocyte/macrophages to an M2 phenotype by the addition of interleukin-4 (IL-4), or whether it is necessary to first pass through an M1 intermediate stage. Retrieved periprosthetic tissues demonstrated increased M1/M2 macrophage ratios compared to non-operated osteoarthritic synovial tissues, using immunohistochemical staining and Western blotting. Uncommitted monocyte/macrophages with/without polymethyl-methacrylate particles were transformed to an M2 phenotype by IL-4 more efficiently when the cells were first passed through an M1 phenotype by exposure to endotoxin. Wear particles induce a pro-inflammatory microenvironment that facilitates osteolysis; these events may potentially be modulated favorably by exposure to IL-4.

Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer

The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR β- and α-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.

Characterization of a New Mouse Model for Peripheral T Cell Lymphoma in Humans

Peripheral T cell lymphomas (PTCLs) are associated with a poor prognosis due to often advanced disease at the time of diagnosis and due to a lack of efficient therapeutic options. Therefore, appropriate animal models of PTCL are vital to improve clinical management of this disease. Here, we describe a monoclonal CD8+ CD4− αβ T cell receptor Vβ2+ CD28+ T cell lymphoma line, termed T8-28. T8-28 cells were isolated from an un-manipulated adult BALB/c mouse housed under standard pathogen-free conditions. T8-28 cells induced terminal malignancy upon adoptive transfer into syngeneic BALB/c mice. Despite intracellular expression of the cytotoxic T cell differentiation marker granzyme B, T8-28 cells appeared to be defective with respect to cytotoxic activity as read-out in vitro. Among the protocols tested, only addition of interleukin 2 in vitro could partially compensate for the in vivo micro-milieu in promoting growth of the T8-28 lymphoma cells.

Interleukin‐7 enhances memory CD8+ T‐cell recall responses in health but its activity is impaired in human immunodeficiency virus infection

Memory CD8(+) T cells regain function during a recall response, but the requirement of signals in addition to antigen during a secondary immune response is unknown. In this study, the ability of interleukin-7 (IL-7) to enhance memory CD8(+ ) CD45RA(- ) CD127(+) T-cell responses in health and in human immunodeficiency virus (HIV) infection was investigated. CD8(+) T-cell-depleted peripheral blood mononuclear cells (PBMCs) from HIV(-) and untreated HIV(+) donors were pulsed with a cytomegalovirus/Epstein-Barr virus/influenza (CEF) peptide pool, and co-cultured with autologous memory CD8(+) T cells in the presence of IL-7. Cell survival and the function of memory CD8(+) T-cell subsets were then evaluated. Memory CD8(+) T-cell proliferation and interferon-γ (IFN-γ) production was enhanced by the presence of antigen, and the addition of IL-7 further enhanced antigen-induced proliferation. In HIV(+) individuals, the presence of antigen enhanced IFN-γ production to a small degree but did not enhance proliferation. Lastly, IL-7 did not enhance antigen-mediated proliferation of memory CD8(+) T cells from HIV(+) individuals. IL-7 therefore appears to have a role in secondary immune responses and its activity is impaired in memory CD8(+) T cells from HIV(+) individuals. These results further our understanding of the signals involved in secondary immune responses, and provide new insight into the loss of CD8(+) T-cell function in HIV infection.

Interleukin-15 attenuates transforming growth factor-beta1-induced myofibroblast differentiation in human fetal lung fibroblasts.

Fibroproliferative diseases are common causes of morbidity and mortality. Interleukin-15 (IL-15) is a pleiotropic cytokine with multiple effects on cells of the immune system. Although IL-15 is also expressed in mesenchymal cells, its effects on the development of fibrosis are unknown. We have previously described an association between serum IL-15 levels and the extent of pulmonary fibrosis in the connective tissue disease systemic sclerosis, suggesting that IL-15 may have profibrotic effects. To test this hypothesis, we studied the effects of IL-15 on myofibroblast differentiation, an in vitro model of fibrosis development. We used human fetal lung fibroblasts for the cytokine stimulation. As a marker of myofibroblast differentiation, α-smooth muscle actin (α-SMA) was analyzed by western blot and quantitative real-time PCR. The well-known profibrotic cytokine, transforming growth factor-β1(TGF-β1), was used for comparison, and TGF-β signaling paths were also studied. IL-15 did not induce α-SMA expression, a marker for myofibroblast differentiation. Unexpectedly, IL-15 counteracted TGF-β1-mediated α-SMA expression. Moreover, TGF-β1-induced expression of collagen, fibronectin and connective tissue growth factor was attenuated by addition of IL-15. There was no effect of IL-15 on early events in the TGF-β signaling cascades. IL-15 has anti-fibrotic properties that, speculatively however, may be insufficient in systemic sclerosis.

Cytokine-mediated Regulation of CX3CL1 in Osteoblasts from Patients with Rheumatoid Arthritis

Introduction CX3CL1 (fractalkine), a membrane-bound chemokine that induces both the adhesion and migration of leukocytes, is involved in the recruitment of cells to tissues undergoing inflammatory responses. To explore the regulation of CX3CL1 in inflammatory bone diseases, we examined CX3CL1 expression in osteoblasts. Methods Human osteoblasts isolated from the femora of rheumatoid arthritis patients were incubated in the presence or absence of various inflammatory stimuli. Culture supernatants were collected, and soluble CX3CL1 levels were determined with an enzyme-linked immunosorbent assay (ELISA). The expression of CX3CL1 mRNA transcripts in osteoblasts was examined using the quantitative TaqMan real-time polymerase chain reaction. Results The combination of tumor necrosis factor (TNF)-α and interferon (IFN)-γ induced dramatic increases in levels of both soluble CX3CL1 protein and mRNA transcripts. CX3CL1 expression in osteoblasts was decreased by the addition of interleukin(IL)-4 or IL-17 but was increased when stimulation by IFN-γ and IL-17 was supplemented with IL-1β In addition, expression was decreased when TNF-α was added. Conclusions Multiple cytokines, including IL-17, are able to either increase or decrease the expression of CX3CL1 by human osteoblasts.

The Effect of Bupivacaine and Morphine in a Coculture Model of Diarthrodial Joints

To perform a controlled laboratory study to evaluate the effect of bupivacaine and morphine on chondrocytes and synovium in a coculture model of diarthrodial joints. A proven coculture model that allows for the assessment of cartilage and synovium exists. The model allows for simulation of the diarthrodial joint in both health and disease by using culture media with or without the addition of interleukin-1. Effects of the presence of bupivacaine and morphine were evaluated by measuring media concentration of glycosamino glycans (GAG), nitric oxide (NO), and prostaglandin E(2) (PGE(2)), and tissue concentration of GAG, water, and collagen. Cell viability was determined through the use of confocal microscopy on days 1 and 2. Cell viability 2 days after exposure to 0.5% bupivacaine was significantly less in the presence of bupivacaine than in the other groups, nearing a 100% decrease in viability. There was little effect of bupivacaine on cartilage water content or the tissue concentration of GAG and collagen. Morphine and bupivacaine both inhibited the expected rise in NO and PGE(2) when interleukin-1 was added to the media. Continuous 0.5% bupivacaine exposure has a clear detrimental effect on chondrocytes in this in vitro study. Both bupivacaine and morphine appear to have anti-inflammatory effects. Continuous morphine exposure does not cause gross chondrotoxicity in vitro and presents itself as a potential alternative intra-articular analgesic. Intra-articular bupivacaine infusion is an effective analgesic strategy and is frequently used in both office and outpatient surgical settings. This study provides evidence that the continued usage of postoperative bupivacaine continuous infusion pumps may have a detrimental effect on chondrocytes. Morphine has been shown to be an effective intra-articular analgesic, and its anti-inflammatory role seen in this study makes it a potential alternative to bupivacaine.

Receptor tyrosine kinases as mediators of injury-induced bradykinin B 1 receptor expression in rabbit aortic smooth muscle

Prolonged in vitro incubation of rabbit aortic rings allows recording contractile responses mediated by the inducible bradykinin B 1 receptors; addition of interleukin (IL)-1 or epidermal growth factor (EGF) to the bathing fluid increases the rate of sensitization, a process partially inhibited by the nonspecific Tyr-kinase inhibitor genistein. The recent development of specific inhibitors for receptor associated Tyr-kinase activities (tyrphostin AG 1478 for EGF receptor, sunitinib for VEGF receptor and others) allows assessing the role of such signaling molecules in this process. AG 1478 reduced the potentiating effects of exogenous EGF, and also the spontaneous sensitization to the agonist des-Arg 9-bradykinin. Sunitinib or GM 6001, a wide spectrum inhibitor of metalloproteinases, had no effect on these responses. In rabbit aortic smooth muscle cells, the cytokines IL-1β and EGF increased the density of binding sites for [ 3H]Lys-des-Arg 9-bradykinin in 4 h; AG 1478 reduced only the effect of exogenous EGF. IL-1 receptor antagonist decreased both the effect of IL-1β and of EGF in rabbit smooth muscle cells. EGF was weakly and slowly coupled to nuclear factor-κB nuclear translocation in these cells, as compared to the effect of IL-1β. EGF-induced EGF receptor autophosphorylation and ERK1/2 phosphorylation was selectively inhibited by AG 1478 in smooth muscle cells; Lys-des-Arg 9-bradykinin did not transactivate EGF receptor in these cells. While the Tyr-kinase activity sensitive to AG 1478 is recruited by tissue damage and exogenous EGF to upregulate bradykinin B 1 receptors in freshly isolated aortas, this signaling system interacts with others (e.g., IL-1) for the optimal expression of B 1 receptors.

Interleukin‐15 stimulates macrophages to activate CD4+ T cells: a role in the pathogenesis of rheumatoid arthritis?

Interleukin-15 (IL-15) is a proinflammatory cytokine that is overexpressed in rheumatoid arthritis (RA), a disease characterized by activation of monocytes/macrophages (MPhi), and by expansion of autoreactive CD4(+) T cells. We hypothesized that IL-15 plays a major role for this expansion of CD4(+) T cells and modulates the phenotype of monocytes/MPhi and their interaction with CD4(+) T cells. Here, we show that IL-15 enhances the proliferation of CD4(+) T cells from patients with RA in peripheral blood mononuclear cell cocultures. To further dissect the underlying mechanisms, we employed MPhi from IL-15(-/-) or IL-15 transgenic mice. These were induced to differentiate or were stimulated with IL-15. Here we show that addition of IL-15 during differentiation of MPhi (into 'IL-15MPhi') and overexpression of IL-15 by MPhi from IL-15(tg) mice leads to increased levels of major histocompatibility complex class II expression. This resulted in enhanced stimulation of antigen-specific CD4(+) T cells in vitro and was accompanied by reduced messenger RNA expression in MPhi for immunosuppressive SOCS3. The proliferation rates of IL-15MPhi and IL-15(tg)MPhi were high, which was reflected by increased p27(Kip1) and reduced p21(Waf1) levels. In view of high serum and synovial levels of IL-15 in patients with RA, our data suggest the possibility that this excess IL-15 in RA may stimulate monocytes/MPhi to activate the characteristic autoreactive CD4(+) T cells in RA.

Induction of Adenovirus Specific T-Cells in Response to 15-Mer Peptides in Cord Blood

Human adenovirus (HAdV) infections are a serious life-threat in haematopoietic stem cell transplantation (HSCT) patients, particularly in children. In recent years, umbilical cord blood transplants (UCBT) emerged as an alternative stem cell source. The advantages are the rapid availability of donor cells, the possibility of using HLA-mismatched transplants and a lower risk of graft versus host disease. However, the immune response after UCBT is often delayed, leading to a higher risk of viral infections. Because no effective antiviral medication exists for severe HAdV infection adoptively transfer of HAdV specific T-cells from cord blood may be a promising treatment. The aim of this study was to see if it was possible to induce HAdV specific T-cells in response to five recently detected 15-mer peptides ( Haveman LM, Int. Imm. 2006), a pool of these five peptides or complete inactivated HAdV (MOI 10). Mitogen Concovalin A ( ConA) was used as control. Cord blood mononuclear cells (CBMC) derived from 34 uncomplicated full term deliveries were isolated and cultured with the different antigens. Proliferation expressed as Stimulation Index (SI) was determined. A SI>1.7 was seen as positive. In 14 neonates a response to in total 36 of the 5 different HAdV peptides could be seen and in 7 of them also a response to the pool of the peptides. Only in 3 neonates a response to complete HAdV could be detected (Figure 1). To induce HAdV specific T-cells CBMCs of 20 neonates were cultured with the different HAdV antigens in addition of interleukin 2 (40IU/ml) for 7 days. By using FACS analysis the upregulation of the activation markers CD25 and CD69 could be detected in response to the HAdV peptides (n=41) or to the complete HAdV (n=26). Induced HAdV specific T-cells expressed the cytotoxins IFN□, TNFα, perforin and granzyme B, produced by CD8+ T-cells, but in higher extent by CD4+ T-cells. This indicates that induced HAdV specific T-cells play an essential role in killing HAdV infections. No clear correlation could be seen between proliferation and the production of the cytotoxins. The induction of a specific immune response to HAdV peptides in cord blood is an important step towards adoptive therapy. [Display omitted]

R455 – Effect of Glucocorticoid on the MUC4 Gene

Problem Among the airway mucin genes, the MUC4 gene is an important gene in its response to inflammatory diseases of the upper airway. However, the expression and regulation of the MUC4 gene in the nasal polyps remains unclear. The purpose of this study was to evaluate the expression of MUC4 mRNA and synthesis of mucin glycoprotein in the nasal polyps before and after treatment with a topical steroid in vivo and in vitro. Methods Nasal polyps were obtained from 20 patients with chronic rhinosinusitis and were subsequently cultured. The level of MUC4 mRNA was measured by reverse-transcription polymerase chain reaction, and the amount of the MUC4 mucin glycoprotein was estimated by the enzyme-linked immunosorbent assay method. Results The expression of MUC4 mRNA was found to be significantly higher in the nasal polyps than in the inferior turbinate (P < .05). The addition of interleukin (IL)-1beta and lipopolysaccharide (LPS) increased the expression of MUC4 mRNA and mucin glycoprotein synthesis in cultured nasal polyp epithelial cells. Treatment with glucocorticoid inhibited the expression of MUC4 mRNA in the nasal polyps; it also inhibited the expression of IL-1beta and LPS-induced MUC4 mRNA and mucin glycoprotein synthesis in cultured nasal polyp epithelial cells. The inhibitory effects of glucocorticoid were restored by treatment with a glucocorticoid receptor antagonist (RU-486). Conclusion These results suggest that the MUC4 gene is expressed in the nasal polyps and that glucocorticoid can control the expression of the MUC4 gene and mucin glycoprotein synthesis. Significance Based on these findings, we conclude that the MUC4 gene is one of the important mucin genes expressed in the nasal polyps and that glucocorticoid can control the expression of MUC4 genes and mucin glycoprotein synthesis.

Effect of Glucocorticoid on the MUC4 Gene in Nasal Polyps

Among the airway mucin genes, the MUC4 gene is an important gene in its response to inflammatory diseases of the upper airway. However, the expression and regulation of the MUC4 gene in the nasal polyps remains unclear. The purpose of this study was to evaluate the expression of MUC4 mRNA and synthesis of mucin glycoprotein in the nasal polyps before and after treatment with a topical steroid in vivo and in vitro. Nasal polyps were obtained from 20 patients with chronic rhinosinusitis and were subsequently cultured. The level of MUC4 mRNA was measured by reverse-transcription polymerase chain reaction, and the amount of the MUC4 mucin glycoprotein was estimated by the enzyme-linked immunosorbent assay method. The expression of MUC4 mRNA was found to be significantly higher in the nasal polyps than in the inferior turbinate (P < .05). The addition of interleukin (IL)-1 beta and lipopolysaccharide (LPS) increased the expression of MUC4 mRNA and mucin glycoprotein synthesis in cultured nasal polyp epithelial cells. Treatment with glucocorticoid inhibited the expression of MUC4 mRNA in the nasal polyps; it also inhibited the expression of IL-1 beta and LPS-induced MUC4 mRNA and mucin glycoprotein synthesis in cultured nasal polyp epithelial cells. The inhibitory effects of glucocorticoid were restored by treatment with a glucocorticoid receptor antagonist (RU-486). These results suggest that the MUC4 gene is expressed in the nasal polyps and that glucocorticoid can control the expression of the MUC4 gene and mucin glycoprotein synthesis.

Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization (3). Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro (3). Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later and excess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20 degrees C.

Interleukin‐1 inhibits firing of serotonergic neurons in the dorsal raphe nucleus and enhances GABAergic inhibitory post‐synaptic potentials

In vitro electrophysiological data suggest that interleukin-1 may promote non-rapid eye movement sleep by inhibiting spontaneous firing of wake-active serotonergic neurons in the dorsal raphe nucleus (DRN). Interleukin-1 enhances GABA inhibitory effects. DRN neurons are under an inhibitory GABAergic control. This study aimed to test the hypothesis that interleukin-1 inhibits DRN serotonergic neurons by potentiating GABAergic inhibitory effects. In vitro intracellular recordings were performed to assess the responses of physiologically and pharmacologically identified DRN serotonergic neurons to rat recombinant interleukin-1beta. Coronal slices containing DRN were obtained from male Sprague-Dawley rats. The impact of interleukin-1 on firing rate and on evoked post-synaptic potentials was determined. Evoked post-synaptic potentials were induced by stimulation with a bipolar electrode placed on the surface of the slice ventrolateral to DRN. Addition of interleukin-1 (25 ng/mL) to the bath perfusate significantly decreased firing rates of DRN serotonergic neurons from 1.3 +/- 0.2 Hz (before administration) to 0.7 +/- 0.2 Hz. Electrical stimulation induced depolarizing evoked post-synaptic potentials in DRN serotonergic neurons. The application of glutamatergic and GABAergic antagonists unmasked two different post-synaptic potential components: a GABAergic evoked inhibitory post-synaptic potentials and a glutamatergic evoked excitatory post-synaptic potentials, respectively. Interleukin-1 increased GABAergic evoked inhibitory post-synaptic potentials amplitudes by 30.3 +/- 3.8% (n = 6) without affecting glutamatergic evoked excitatory post-synaptic potentials. These results support the hypothesis that interleukin-1 inhibitory effects on DRN serotonergic neurons are mediated by an interleukin-1-induced potentiation of evoked GABAergic inhibitory responses.

Differential Effect of Burn Injury on Fibroblasts from Wounds and Normal Skin

Although there are well-recognized fluctuations in the systemic concentration of cytokines and growth factors after burn injuries, the effect on wound-healing potential in patients is not well understood. The objective of this study was to characterize the proliferation rate and response of wound and dermal fibroblasts to cytokines in burn patients compared with normal subjects. Polyvinyl alcohol sponges were implanted subcutaneously in normal subjects and burn patients soon after admission. Sponges were removed for wound fibroblast explantation after 12 days. At the same time, a small piece of skin was excised for dermal fibroblast explantation. Fibroblast proliferation was then quantified after exposure to 10% fetal bovine serum, 1% fetal bovine serum, interleukin-1, transforming growth factor-beta1, or interferon-alpha2b. Normal subjects' dermal fibroblasts (n = 7) exposed to 10% fetal bovine serum showed significantly increased proliferation relative to normal subjects' wound fibroblasts (n = 3) (p < 0.0005), burn patients' dermal fibroblasts (n = 5) (p < 0.05), and burn patients' wound fibroblasts (n = 5) (p < 0.0001). Burn patients' dermal fibroblast proliferation was also significantly augmented relative to burn patients' wound fibroblasts (p < 0.005); however, there was no significant difference between the two wound fibroblast types. Proliferation of burn subjects' fibroblasts was significantly enhanced with the addition of interleukin-1 and significantly decreased for dermal fibroblasts with interferon-alpha2b. A significant elevation of proliferation with transforming growth factor-beta1 was seen only with burn patients' dermal fibroblasts. The data suggest that systemic mediators markedly alter the proliferation potential of dermal fibroblasts but not of wound fibroblasts. However, the wound environment substantially alters both the proliferation rate and the responsiveness of fibroblasts to cytokines. Thus, the data support the value of using wound fibroblasts during preliminary in vitro experiments to investigate wound-healing modification by cytokine manipulation.

Interleukin-15 (IL-15) enhances, as well as increases the sensitivity of human CD8+ memory T cell subsets to stimulation by the pro-inflammatory cytokines IL-12 and IL-18 (95.3)

Abstract Human memory CD8+ T cell subsets, termed central memory and effector memory, can be identified by expression of CD45RA, CD62L, and CCR7. Accordingly, functional differences have been described for each subset, reflecting unique roles in immunological memory. The common gamma chain cytokines IL-15 and IL-7 can induce proliferation and differentiation of human CD8+ T cell subsets, as well as increased effector functions. Here, we observed that addition of IL-15 or IL-7 to cultures of human CD8+ T cells profoundly enhanced the IL-12+IL-18 pathway of IFN-γ production. Importantly, IL-15 and IL-7 lowered the threshold concentrations of IL-12 and IL-18 required for induction of IFN-γ by 100-fold. Comparison of IL-15 and IL-7 demonstrated that IL-15 was superior for its ability to enhance IL-12+IL-18-induced IFN-γ, without evidence of synergy between IL-15 and IL-7. We also observed that IL-15 and IL-7-mediated enhancement of IL-12+IL-18-induced IFN-γ production was a functional property of effector-memory CD8+ T cells. Despite a lack of association between cell division and IFN-γ, loss of CD62L expression correlated well with acquisition of IL-12+IL-18-induced IFN-γ. Indeed, purified central memory T cells stimulated with IL-15 and IL-7 developed into effector memory T cells with potent IL-12+IL-18-induced IFN-γ. Thus, in addition to its known role in development of T cell memory, IL-15 may amplify CD8+ T cell effector functions by increasing sensitivity to pro-inflammatory cytokine stimulation. This research was supported by a grant from the Jeffress Memorial Trust Fund and the Department of Microbiology at VCU.

The effect of age on the cognate function of CD4+ T cells

With increasing age, the ability to produce protective antibodies in response to immunization declines, resulting in reduced efficacy of vaccination. We have examined how reductions in CD4(+) T-cell function contribute to reduced humoral responses, using a model that allows us to compare identical numbers of antigen-specific naive T cells from young and aged T-cell receptor transgenic mice. Naive cells from aged mice exhibit reduced responses, both in vitro and in vivo. In vitro, responses of aged T cells can be enhanced by addition of interleukin (IL)-2. In vivo, using an adoptive transfer model with young hosts, naive cells from aged mice exhibit significant reductions in cognate helper function, leading to reduced B-cell expansion and differentiation. These age-related defects could be overcome by prior in vitro T helper 2 effector generation with aged T cells. This improvement in cognate function of the aged effectors may be related to the enhancement of CD154 expression, which occurs on aged T cells in the presence of exogenous IL-2. We also found no difference in B-cell expansion and differentiation when young cells were transferred to young or aged hosts. Our results indicate that age-related reductions in humoral responses are mainly due to defects in the cognate helper function of naive CD4(+) T cells from aged individuals.

Large-Scale Preparation of Human Anti—Third-Party Veto Cytotoxic T Lymphocytes Depleted of Graft-Versus-Host Reactivity: A New Source for Graft Facilitating Cells in Bone Marrow Transplantation

Induction of donor type chimerism in mildly prepared hosts without graft-versus-host disease (GvHD) is a most desirable goal in bone morrow transplantation. We have recently demonstrated in a mouse model that donor veto cytotoxic T lymphocytes (CTLs) can facilitate the induction of donor type chimerism in sublethally irradiated recipients without causing GvHD if they are effectively depleted of alloreactivity against host cells by means of stimulation against a third party. We extend this approach to human cells, by preparing CTLs in two major steps: primary culture in the absence of interleukin 2, leading to death by neglect of antihost clones, and addition of interleukin 2 and subsequent dilution of antihost clones as a consequence of the expansion of the anti-third-party clones. CTLs prepared in this way specifically suppress host cytotoxic T cells directed against antigens of the donor, but not against fourth-party antigens, as demonstrated in a standard 51Cr release assay. We conclude that human anti-third-party CTLs afford a new source of veto cells that are depleted of potential graft-versus-host-reactive clones. The cells generated by this approach could potentially be used to facilitate engraftment of allogeneic hematopoietic stem cells.