Expansion of invariant natural killer T cells from systemic lupus erythematosus patients by alpha-Galactosylceramide and IL-15.

CD1d-restricted invariant natural killer T cells (iNKT cells) may play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Interleukin (IL)-15 is a pro-inflammatory cytokine which is over-expressed in SLE patients. In the present study, we investigated the iNKT cell expansion of mononuclear cells (MNCs) from SLE patients following 10 days' culture with α-galactosylceramide (α-Galcer) and /or IL-15. We sought to determine the phenotypic and functional characteristics of the expanded iNKT cells compared to healthy controls and correlated with disease activity. We observed that 1. The percentages of Vα24+/Vß11+ iNKT cells following 10-day incubation was lower in SLE groups compared to controls; 2. The percentages and absolute numbers of Vα24+/Vß11+ iNKT cells were expanded by α-galactosylceramide (α-Galcer), and further enhanced with IL-15 in SLE patient, but the effect of IL-15 was much lower than controls; 3.IL-15 +α-Galcer expanded CD3+/CD56+ NKT-like cells from SLE patients, especially with active disease 4. The CD161+ Vα24+/Vß11+ iNKT cells in SLE were more responsive to α-Galcer stimulation than the CD161- counterpart; 5. IL-15 decreased apoptosis of α-Galcer activated SLE iNKT cells; 6. IL-15 enhanced CD69, CD1d and CD11a expression on α-Galcer treated iNKT cells; 7. The IL-4 production of iNKT cells was decreased in SLE patients compared to controls; 8. IL-15 increased IFN-γ and IL-4 production of SLE iNKT cells; 8. IL-15 failed to augment the ability of iNKT cells to aid NK-mediated K562 cytolysis in SLE patients; 9. CD161 positivity, granzyme B and perforin expression of α-Galcer+IL-15 expanded iNKT cells correlated with C3 levels in SLE patients. Taken together, our results demonstrated numeric and functional deficiency of iNKT cells and their response to IL-15 in SLE patients. Our finding may provide insight for using adoptive iNKT cell therapy in autoimmune diseases.

Phenotypic and functional characterization of natural killer cells in rheumatoid arthritis-regulation with interleukin-15.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial inflammation and joint destruction. Previous studies have shown that natural killer (NK) cells may play an important role in the pathogenesis of RA. Interleukin (IL)-15, a pro-inflammatory cytokine which induces proliferation and differentiation of NK cells, is overexpressed in RA. In this present study, we examine various NKRs and adhesion molecule expression on NK cells from RA patients and their response to IL-15 stimulation. We also sought to study cytokine-induced memory-like (CIML) NK cells in RA patients. We established that 1. RA patients had higher NK cell percentages in peripheral blood and their serum IL-15 levels were higher compared to healthy volunteers; 2. NK cells from RA patients showed lower NKp46 expression and an impaired CD69 response to IL-15; 3. NK cells from RA patients showed higher CD158b and CD158e expression but lower CD62L expression; 4. exogenous IL-15 up-regulated CD69, CD158b, CD158e but down-regulated NKp46 and CD62L expression in RA; 5. As to CIML NK cells, restimulation - induced NK cytotoxicity and IFN-γ production was impaired in RA patients, 6. Reduced NKp46, perforin, and granzyme B expression on NK cells was found in RA patients with bone deformity and erosion, 7. RA disease activity (DAS28) showed inverse correlation with the percentages of CD56+CD3- NK cells, and NKp46 and perforin expression on NK cells, respectively. Taken together, our study demonstrated differential expression of various NK receptors in RA patients. NKp46, CD158e, and perforin expression on NK cells may serve as markers of RA severity.

Cytotoxic Function and Cytokine Production of Natural Killer Cells and Natural Killer T-Like Cells in Systemic Lupus Erythematosis Regulation with Interleukin-15.

Natural killer cells and NKT-like cells are the first line immune defense against tumor and virus infection. Deficient NK and NKT-like cell effector function may contribute to increased susceptibility to infection in SLE patients. We sought to examine the perforin and granzyme B expression, interferon-gamma (IFN-γ), and tumor-necrosis factor-alpha (TNF-α) production and CD107a degranulation of NK and NKT-like cells from SLE patients and their regulation by IL-15. We established that (1) perforin expression on SLE NK cells was decreased but unrelated to disease activity; (2) the MFI of granzyme B was increased in NK cells from SLE patients with active disease, associated with increased percentages of granzyme B+ CD56bright NK cells; (3) NK cells from active SLE patients, both CD56dim and CD56bright NK subsets, produced higher IFN-γ compared to controls; (4) CD56dim, but not CD56bright NK cells from active SLE patients, produced lower TNF-α, compared to inactive SLE patients and controls; (5) CD107a degranulation of SLE NK cells was comparable to controls; (6) IL-15 enhanced perforin/granzyme B expression, IFN-γ/TNF-α production, and CD107a degranulation of NK cells from SLE patients; and (7) similar observations were found for CD56+CD3+ NKT-like cells. Taken together, we demonstrated the differential expression of the heightened granzyme B and decreased TNF-α in NK and NKT-like cells in SLE patients. Higher granzyme B expression of NK and NKT-like cells in active SLE patients, further enhanced by circulating IL-15, may contribute to the maintenance of inflammation in SLE.

Activating and inhibitory receptors on natural killer cells in patients with systemic lupus erythematosis-regulation with interleukin-15.

Natural killer (NK) cells may play an important role in the pathogenesis of SLE. Interleukin(IL)-15, an NK-enhancing cytokine, is over-expressed in SLE patients. In the present study, we examined the effect of IL-15 on NK cytotoxicity of SLE patients, and the expression of various activating and inhibitory NK receptors on NK cells from SLE patients in relation to disease activity. We also sought to determine how IL-15 would affect the NK receptor expression on NK cells from SLE patients. PBMCs were collected from 88 SLE patients with inactive disease activity (SLEDAI score<6) and active disease activity (SLEDAI score≥6), 26 age-matched healthy adults were used as controls. PBMC were incubated in the presence or absence of IL-15 (10ng/ml) for eighteen hours. CD3-CD56+ lymphoctes were gated using flow cytometry and further divided into CD56dim and CD56bright subsets according to the MFI of CD56. We observed that 1. Serum IL-15 was elevated in SLE patients, and higher in active disease than in inactive disease; 2. NK cytotoxicity of SLE patients was deficient compared to controls and showed an impaired response to IL-15 compared to controls; 3.CD69, CD94, NKG2A, NKp30, and CD158b on NK cells from SLE patients were higher than controls, and could be further enhanced by IL-15; 4. NKp46 expression from SLE patients was higher than controls, but down-regulated by IL-15; 5.Deficient NKG2D and NKAT-2 expression were found on NK cells from SLE patients, which were enhanced by IL-15; 6. A unique NKp46- subset and CD158b+ subsets were observed in NK cells from SLE patients but not controls. 7. Unlike controls, CD158k on NK cells from SLE patients failed to respond to IL-15. Taken together, we demonstrated the aberrant NCR and iNKR expression on NK cells and their distinct response to IL-15 in SLE patients. As IL-15 predominantly aggravates the aberrant NKR expression found in SLE, IL-15 antagonist may have therapeutic benefits in SLE patients.

Azithromycin modulates immune response of human monocyte-derived dendritic cells and CD4+ T cells.

Azithromycin (AZM) is a macrolide antibiotic that exhibits anti-inflammatory activity aside from its antimicrobial effect, a feature that may ameliorate certain inflammatory disorders and prevent graft-versus-host disease in patients receiving stem cell transplantation. In the present study, we investigated the ability of AZM to influence the function of human monocyte-derived dendritic cells (DCs) and CD4+ T cells. We found that AZM down-regulated CD80, CD86, and HLA-DR expression in lipopolysaccharide (LPS)-stimulated DCs and suppressed interleukin (IL)-6, IL-10, IL-12, and tumor necrosis factor-alpha production in these cells. In addition, AZM increased endocytosis and/or expression of Toll-like receptor (TLR)2, TLR4, and TLR9 in DCs and suppressed anti-CD3/CD28-induced CD4+ T cell proliferation and interferon-gamma production, an effect that was synergistic with dexamethasone. Finally, AZM suppressed DC-induced allogeneic T cell proliferation and cytokine production. Our study demonstrates that AZM modulates DC and CD4+ T cell function and may be of therapeutic benefit in various inflammatory disorders.

Effect of Interleukin-15 on CD11b, CD54, and CD62L Expression on Natural Killer Cell and Natural Killer T-Like Cells in Systemic Lupus Erythematosus.

Adhesion molecules may play an important role in systemic lupus erythematosus (SLE) pathogenesis. We investigated the effect of interleukin- (IL-) 15 on CD11b, CD54, and CD62L expression on natural killer (NK) cells, T cells, and CD56+CD3+ NKT-like cells from SLE subjects and healthy controls. SLE patients had decreased circulating NK cells and NKT-like cells compared to controls. NK cells from SLE patients showed higher CD11b and CD62L expression compared to controls. IL-15 enhanced CD11b and CD54 but downregulated CD62L expression on NK cells from SLE patients. Similar observations were found for T cells and NKT-like cells. NK cells from SLE patients expressed higher CD56 than controls; both could be further enhanced by IL-15. IL-15 also enhanced CD56 expression of NKT-like cells from SLE patients. A greater degree of IL-15 induced downregulation of CD62L on NKT-like cells noted in SLE patients compared to controls. The percentage of CD11b expressing NK cells and the % inhibition of CD62L expression on NKT-like cells by IL-15 correlated with serum anti-dsDNA levels in SLE patients, respectively. Taken together, we demonstrated the dysfunctional NK and NKT-like cells in SLE patients with regard to CD11b and CD62L expression and their response to IL-15.

Interleukin-15 enhances the expansion and function of natural killer T cells from adult peripheral and umbilical cord blood.

Invariant natural killer T cells (iNKT cells) are innate-like non-conventional T cells restricted by the CD1d molecule that are unique in their ability to play a pivotal role in immune regulation. Deficient iNKT function has been reported in patients receiving umbilical cord blood (UCB) transplantation. We sought to determine the effect of interleukin (IL)-15 on α-galactosylceramide (α-GalCer)-expanded iNKT cell function from UCB and adult peripheral blood (APB) mononuclear cells (MNCs). Fresh APB and UCB MNCs were cultured with IL-15 (50 ng/ml) in the presence or absence of α-GalCer (100 ng/ml) for 10 days. Cells were harvested for examination of cell yield, apoptosis, cytokine production and cytotoxic function of Vα24(+)/Vß11(+) iNKT cells. We observed that α-GalCer-expanded APB and UCB iNKT cells and such expansion was further enhanced with IL-15. The percentage of CD3(+)CD56(+) NKT-like cells in both APB and UCB MNCs was increased with IL-15 but not with α-GalCer. Apoptosis of UCB iNKT cells was ameliorated by IL-15. Although APB and UCB iNKT cells secreted lower IFN-γ, it could be enhanced with IL-15. The expression of perforin in APB iNKT cells can also be enhanced with IL-15. UCB Vα24(+)Vß11(+) iNKT cells further augmented K562 cytotoxicity mediated by IL-15. Taken together, these results demonstrated the relative functional deficiencies of α-GalCer induced UCB iNKT cells, which can be ameliorated by IL-15. Our findings suggest a therapeutic benefit of IL-15 immunotherapy during the post-UCB transplant period when iNKT function remains poor.

Cytokine activation of natural killer cells.

CD3(-)CD56(+) natural killer (NK) cells can kill various tumors in a non major histocompatibility complex (MHC)-restricted fashion. Recent advances have been made in the application of NK cells for the treatment of patients with acute myelogenous leukemia (AML). Allogeneic donor-derived NK cells can be activated in vitro and infused into patients receiving stem cell transplants. We describe in this chapter the method to activate NK cells with cytokines and to ascertain their degree of activation.

Expansion of regulatory T cells from umbilical cord blood and adult peripheral blood CD4(+)CD25 (+) T cells.

CD4(+)CD25(+) regulatory T cells (Treg), if properly expanded from umbilical cord blood (UCB), may provide a promising immunotherapeutic tool. Our previous data demonstrated that UCB CD4(+)CD25(+) T cells with 4-day stimulation have comparable phenotypes and suppressive function to that of adult peripheral blood (APB) CD4(+)CD25(+) T cells. We further examined whether 2-week culture would achieve higher expansion levels of Tregs. UCB CD4(+)CD25(+) T cells and their APB counterparts were stimulated with anti-CD3/anti-CD28 in the presence of IL-2 or IL-15 for 2 weeks. The cell proliferation and forkhead box P3 (FoxP3) expression were examined. The function of the expanded cells was then investigated by suppressive assay. IL-21 was applied to study whether it counteracts the function of UCB and APB CD4(+)CD25(+) T cells. The results indicate that UCB CD4(+)CD25(+) T cells expanded much better than their APB counterparts. IL-2 was superior to expand UCB and APB Tregs for 2 weeks than IL-15. FoxP3 expression which peaked on Day 10-14 was comparable. Most importantly, expanded UCB Tregs showed greater suppressive function in allogeneic mixed lymphocyte reaction. The addition of IL-21, however, counteracted the suppressive function of expanded UCB and APB Tregs. The results support using UCB as a source of Treg cells.

Regulation of CD28 expression on umbilical cord blood and adult peripheral blood CD8+ T cells by interleukin(IL)-15/IL-21.

Interleukin (IL)-15 and IL-21, both belonging to common γ-chain-signaling cytokine family, have an important role to maintain homeostatic proliferation of CD8(+) T cells. CD28, an essential co-stimulatory molecule on T cells, may be a marker of replicative senescence. We investigated the effect of IL-15 and IL-21, alone or in combination, on activation, apoptosis, cytokine production and cytotoxic function of magnetic bead purified umbilical cord blood (UCB) and adult peripheral blood (APB) CD8(+) T cells with regards to their CD28 expression. We established that (1) IL-15-induced CD8(+) T cell proliferation was associated with a preferential expansion of CD28(-) population in UCB, which could be partially counteracted by IL-21; (2) UCB CD8(+) T cells were more readily responsive to IL-15 compared to their adult counterparts in terms of CD69 expression, with the majority of CD69-bearing CD8(+) T cells were CD28(-); (3) IL-21 further promoted interferon-gamma, but not tumor necrosis factor-alpha production from IL-15 treated CD8(+) T cells; (4) IL-21 also synergized with IL-15 to enhance perforin and granzyme B expression of CD8(+) T cells, especially in APB CD8(+)CD28(-) subsets; (5) IL-21 resulted in CD8(+) T cells apoptosis both in APB and UCB cells, mainly in CD8(+)CD28(-) subsets. Taken together, we demonstrate differential IL-15/IL-21 response in UCB CD8(+) T cells with regards to CD28 expression. Our results suggest that combining IL-21 and IL-15 immunotherapy may be better than IL-15 alone to ameliorate graft-versus-host disease while preserving antitumor effect in the post-UCB transplantation period.

Effect of influenza A infection on umbilical cord blood natural killer function regulation with interleukin-15.

BACKGROUND: Influenza A is a major pathogen of humans and has the potential to cause worldwide pandemics. Natural killer (NK) cells are important effector cells in the innate immune response against viruses, including influenza A. Infants are more susceptible to severe influenza A viral infection, possibly attributed in part to their defective NK function. METHODS: We compared the NK responses to influenza using umbilical cord blood (UCB) and adult peripheral blood (APB) mononuclear cells and purified NK cells. RESULTS: Influenza A induced dose-dependent apoptosis of NK cells with down-regulation of NKp46 expression, which was more pronounced in UCB. Both UCB and APB NK cells responded to influenza infection by up-regulating CD69 and CD107a expression, a process further enhanced by interleukin (IL) 15. Influenza exposure also down-regulated perforin expression and K562 cytotoxicity in UCB NK cells, which was partially restored by IL-15. The production of interferon (IFN) γ and tumor necrosis factor (TNF) α by NK cells in responding to influenza was further enhanced by IL-15. CONCLUSIONS: Our findings show differential NK responses between newborns and adults. IL-15 may be beneficial in combating influenza by enhancing cytotoxic function and IFN-γ production.

Umbilical cord blood immunology: relevance to stem cell transplantation.

Because of its easier accessibility and less severe graft-versus-host disease, umbilical cord blood (UCB) has been increasingly used as an alternative to bone marrow for hematopoietic stem cell transplantation. Naiveté of UCB lymphocytes, however, results in delayed immune reconstitution and infection-related mortality in transplant recipients. This review updates the phenotypic and functional deficiencies of various immune cell populations in UCB compared with their adult counterparts and discusses clinical implications and possible therapeutic strategies to improve the outcome of stem cell transplantation.

Effect of interleukin-15 on alloreactivity in umbilical cord blood.

Interleukin(IL)-15 is a promising immunotherapeutic agent for immune reconstitution following stem cell transplantation. To investigate whether IL-15 would aggravate graft-versus-host disease (GVHD) in the setting of unrelated umbilical cord blood (CB) transplantation, we examined the effect of IL-15 on activation marker expression, proliferation and cytokine production of CB in a one-way mixed lymphocyte culture (MLC) assay. We found that IL-15 differentially enhanced CD69 and CD25 expression on CB T cells following allo-stimulation. The maximum degree of allo-specific CB proliferation was achieved on Day 6. IL-15 down-regulated the CB alloreactive proliferative response on Days 4, 6, and 8, with preferentially enhanced autologous proliferation. Exogenous IL-15 further enhanced CB TNF-alpha and IL-10 production in both autologous and allogeneic MLC 6 days after allopriming. Thus, IL-15 was effective in enhancing activation marker expression and cytokine production during CB alloreactivity, but failed to enhance allospecific proliferation. Further studies would be needed to study the role of IL-15 on GVHD in the setting of CB transplantation.