Stat5b Is Essential for Natural Killer Cell–mediated Proliferation and Cytolytic Activity

We have analyzed the immune system in Stat5-deficient mice. Although Stat5a^−/− splenocytes have a partial defect in anti-CD3-induced proliferation that can be overcome by high dose interleukin (IL)-2, we now demonstrate that defective proliferation in Stat5b^−/− splenocytes cannot be corrected by this treatment. Interestingly, this finding may be at least partially explained by diminished expression of the IL-2 receptor β chain (IL-2Rβ), which is a component of the receptors for both IL-2 and IL-15, although other defects may also exist. Similar to the defect in proliferation in activated splenocytes, freshly isolated splenocytes from Stat5b^−/− mice exhibited greatly diminished proliferation in response to IL-2 and IL-15. This results from both a decrease in the number and responsiveness of natural killer (NK) cells. Corresponding to the diminished proliferation, basal as well as IL-2– and IL-15–mediated boosting of NK cytolytic activity was also greatly diminished. These data indicate an essential nonredundant role for Stat5b for potent NK cell–mediated proliferation and cytolytic activity.     

Characterization of human killer cell reactivity against porcine target cells: differential modulation by cytokines

Abstract: The cytotoxic cell response to porcine cells by human lymphocytes, and the modulation of cytolytic cellular activity by human cytokines were investigated. Human peripheral blood mononuclear cells (PBMC) and purified lymphocyte subsets were co-cultured with fresh irradiated porcine stimulator cells and examined for the development of lytic activity and for their proliferative response. Porcine target cells included a new cell line, MS-PBMC-J2 (designated J2; SLA-DR⁺MHC class I⁺CD2⁺CD3^–CD8⁺CD16⁺CD45⁺), aortic and microvascular endothelial cells. Initial results showed that natural killer (NK) cells were fivefold more efficient in killing porcine target cells compared with T cells. IL-12 augmented the killing of porcine target cells by human NK cells beyond that induced by stimulation with cells alone. In contrast, IL-2 and IL-15 often induced substantial human NK cell mediated killing of porcine target cells, including endothelial cells in the case of IL-2 where such targets were examined, even in the absence of stimulator cells. Finally, neither IL-18 nor IL-8 had any effect beyond background on NK cell mediated killing of porcine target cells. These findings show that cytokines that would be produced in a xenograft setting clearly modulate the ability of human cytolytic cells to kill porcine targets. In addition, fresh unstimulated human NK cells lysed J2 and porcine aortic endothelial cells, but not porcine microvascular endothelial cells, suggesting the possibility of rapid attack of xenografts by NK cells, and differential susceptibility of endothelial cells from different vascular structures to this attack.     

IL-12 administered in vivo to young and aged mice. Discrepancy between the effects on tumor growth in vivo and cytotoxic T lymphocyte generation ex vivo: dependence on IFN-{gamma}

Because IL-12 restores allogeneic cytotoxic T lymphocyte (CTL)activity by T cells of aged mice in vitro, we initially assessedwhether IL-12 could overcome age-related deficits when givento aged mice in vivo. Growth of P815 (H-2^d) was enhanced inaged compared with young BALB/c (H-2^d) mice and tumor growthwas curtailed by IL-12 in both age groups. Unexpectedly, secondaryCTL stimulated ex vivo with P815 were reduced in IL-12-treatedmice compared with controls. Primary CTL generated ex vivo acrossMHC differences in IL-12-treated BALB/c and C57BL/6 young micewere reduced by 90–99%, were dose- and time-dependent,and were associated with reduced allo-stimulated NK-like activityand [³H]thymidine incorporation. IFN- was elevated in sera andin supernatants from allo-stimulated cultures from IL-12-treatedmice, while IL-4 was reduced in such supernatants, suggestingthat, despite reduced CTL, IL-12 was associated with increasedT_h1- and reduced T_h2-type cytokine production. IL-12 also inducedsplenomegaly, primarily due to increased numbers of cells lackingmarkers of mature T, B and NK cells, or macrophages, or polymorphonuclearleukocyte morphology. IFN- mutant mice exhibited reduced splenicenlargement in response to IL-12, suggesting that the splenomegalywas due, in part, to IFN- production. However, reduced CTL generationwas not due entirely to dilution of CTL precursor cells becausespleen cellularity and size increased 3-fold while CTL activitydecreased 10- to 100-fold, and CTL generation normalized toCD8⁺ T effector cells was still significantly reduced in IL-12-treatedmice. Interestingly, purified CD4⁺ and CD8⁺ T cells from IL-12-treatednormal mice exhibited greater proliferative and cytolytic activitiesrespectively compared with controls. Thus, effector T cellsin IL-12-treated mice were not impaired, but exhibited augmentedresponsiveness, suggesting that IL-12 induced complex interactionsamong spleen cell populations and that these effects, in part,are mediated by IFN-.     

Lipophilic statins suppress cytotoxicity by freshly isolated natural killer cells through modulation of granule exocytosis

NK cells, a component of the innate immune system, provide a first line of defense against viral infections and malignancies, interact with the adaptive immune system and have a role in rejection of allogeneic bone marrow transplants and solid allo- and xenotransplants. Immunoregulatory activity by the anti-hypercholesterolemia agents, 3-hydroxy-3-methyl-glutaryl Coenzyme A (HMG-CoA) reductase inhibitors, known as statins, has recently been reported. We analyzed the effects of three statins on human NK cell cytotoxicity. Two lipophilic statins (simvastatin and fluvastatin) suppressed the cytotoxic activity of fresh and IL-2-stimulated NK cells, while pravastatin, a hydrophilic statin, did not. Suppression was not associated with changes in intracellular perforin, granzyme A or granzyme B levels, or with changes in expression of leukocyte function-associated antigen-1, an integrin known to regulate NK activity and reported to be altered by statin treatment. Decreased cytotoxicity was associated with decreased CD107a surface expression, indicating that the exocytosis pathway was compromised by simvastatin and fluvastatin but not by pravastatin. Mevalonate, the immediate downstream product of HMG-CoA reductase, partially reversed the effect of lipophilic statins on cytotoxicity and CD107a expression. Lipophilic statins also suppressed the release of the granule component, granzyme B, by IL-2-activated NK cells following stimulation with K562. That lipophilic statins suppress NK cell activity through inhibition of the exocytosis pathway suggest an additional potential role for statins in inhibition of transplantation responses.     

Could heme-oxygenase-1 have a role in modulating the recipient immune response to embryonic stem cells?

Pluripotent human embryonic stem cells (hESCs) may provide a potential source of cellular therapies, but as allogeneic cells may require evading the recipient's immune response. Using an NIH-registry hESC line, it was found that undifferentiated hESCs induce a reduced proliferative response compared to PBMC and demonstrate that this diminished response correlates with the activity of heme oxygenase-1 (HO-1). Inhibition of HO-1 significantly increases T cell proliferation against hESC, indicating the potential suppression of these cells during transplantation of allogeneic hESC. These data suggest the hypothesis that HO-1 provides a mechanism for protecting hESCs in vivo.     

Human NK cells can lyse porcine endothelial cells independent of their expression of Galalpha(1,3)-Gal and killing is enhanced by activation of either effector or target cells

Abstract: Background: Xenotransplantation of pig organs may provide an approach to alleviate the severe shortage of human organs. Natural antibodies against Galα(1,3)‐Gal (αGal) epitopes cause hyperacute rejection of pig organs in primates. However, evidence for the role of αGal in the natural killer (NK) cell‐mediated xenoresponse has been contradictory. Methods: We investigated the recognition of αGal by human NK cells using endo‐β‐galactosidase C, an enzyme that cleaves αGal, and endothelial cells (EC) from α1,3‐galactosyltransferase null pigs that do not synthesize αGal. Endo‐β‐galactosidase C treatment variably reduced the susceptibility of porcine EC to lysis by fresh human NK cells. Results: Removal of αGal from porcine EC using endo‐β‐galactosidase C, produced variable results, i.e. cytotoxicity was decreased in half of the human NK cell donors tested. The two EC strains from αGal?/? pigs were marginally, and not significantly, less susceptible to lysis by naïve human NK cells compared with αGal‐expressing cells obtained from animals from the same herd, but these differences were not statistically significant (P > 0.10). Treatment of porcine EC with recombinant human tumor necrosis factor (TNF)‐α, which is known to activate porcine EC, enhanced the susceptibility of all target cells to lysis by fresh human NK cells. Surface expression of MHC or adhesion molecules on αGal?/? cells, compared with wild type cells, showed no consistent difference in either MHC or adhesion molecules CD106 (VCAM‐1), CD31 (PECAM) or CD62E (E‐selectin), either with or without TNF‐α stimulation, that could explain the differential susceptibility to lysis. Strikingly, all αGal?/? and wild type EC exhibited similar susceptibility to human NK cells that had been cultured for 5 days with or without interleukin‐2. Conclusions: These findings demonstrate that human NK cells can kill porcine targets in the absence of αGal, and donor variability plays a major role in whether αGal has a role in determining susceptibility of porcine EC to lysis. Moreover, susceptibility to lysis of αGal null EC is enhanced to the level of wild type EC by activation of either effector or target cells. Elimination of αGal alone from source pigs will be insufficient to circumvent the NK cell mediated destruction of porcine EC.