Characterization of baboon NK cells and their xenogeneic activity.

BACKGROUND: Baboons are commonly used as models for transplantation and preclinical testing of various types of therapeutic agents. For proper assessment of information gathered from these models, differences between the baboon and human immune systems need to be characterized. Natural killer (NK) cells are the first line of defense against many infectious agents and cancer and are important mediators of transplantation rejection reactions, particularly during xenotransplantation. In this study, we examined baboon NK cell function and developed methods for purifying and expanding these cells. METHODS: Baboon NK cells were analyzed using a combination of extracellular and intracellular cell staining, cell sorting, interleukin (IL)-2 mediated stimulation and expansion, and 4 h cytotoxicity assays with human and pig target cell lines. RESULTS: Baboon peripheral blood mononuclear cell (PBMC) exert very low but detectable cytolytic activity against both human (K562) and pig (PAEC, J2) target cells, and this activity is enhanced within 4 h of treatment with IL-2. Like human NK cells, many baboon PBMC express the lytic enzymes granzyme A, granzyme B, and perforin. Based on these markers, we identified a subpopulation of CD3(-) baboon lymphocytes that are CD8(dim) and CD16(bright) that likely represents the baboon NK cells. These cells also are characterized by expression of the natural cytotoxicity receptor NKp46. Baboon CD3(-)NKp46(+) cells purified by flow cytometric cell sorting have high cytolytic capacity that can be further enhanced by IL-2 stimulation. These baboon NK cells can be expanded in vitro and retain extremely high cytolytic capacity. While fresh baboon lymphocytes express very little CD56, the expanded baboon NK cells are predominantly CD56(+); approximately 10% of the expanded NK cells are CD56(dim), and the remainder are CD56(bright). CONCLUSIONS: Baboon NK cells that are IL-2 responsive can be identified on the basis of a CD3(-)NKp46(+)CD8(dim)CD16(+/-) or CD3(-)CD8(dim)CD16(bright) phenotype and can be isolated and expanded in culture. These results may allow for a more accurate representation of the human innate immune system in baboon models and more accurate analyses of the role of the baboon innate immune system cells in preclinical models.

Characterization and expansion of baboon CD4+CD25+ Treg cells for potential use in a non-human primate xenotransplantation model.

BACKGROUND: It is well established that CD4(+)CD25(+) regulatory T (Treg) cells can modulate allogeneic immune responses. Xenotransplantation, proposed as a means to address the critical shortage of human organs, may also benefit from similar approaches to avert rejection. Baboons are a preferred preclinical animal model for xenogeneic organ transplantation experiments, and the characterization of baboon Treg cells will be beneficial to future tolerance studies in this animal model. METHODS: We analyzed CD4(+)CD25(+) T cells from baboon lymph nodes, spleens, and blood by flow cytometry, then purified and expanded porcine antigen-specific baboon CD4(+)CD25(high) cells in vitro to evaluate their regulatory activity in the baboon anti-pig xenogeneic responses. RESULTS: CD4(+)CD25(high) T cells were 1.7%, 3.1%, and 1.9% of baboon splenic, lymph node, and blood T cells, respectively. The CD4(+)CD25(high) T cells expressed the Treg cell-associated transcription factor, FoxP3. Proliferation/suppression assays using irradiated pig peripheral blood mononuclear cells as stimulators showed that Treg cells suppressed the vigorous baboon CD4(+)CD25(-) T-cell anti-pig proliferation response and cytokine secretion. Expanded baboon Treg cells suppressed baboon anti-pig CD4(+)CD25(-) T-cell proliferation approximately 4- to 10-fold more than freshly isolated Treg cells. Expanded Treg cells suppressed proliferation to primary cells from the same pig used for expansion more effectively than proliferation to stimulators from a different strain of pig, suggesting a level of antigen specificity. CONCLUSION: We demonstrate that baboon Treg cells suppress immune responses to xenogeneic stimulation. These studies suggest that adoptive transfer of expanded Treg cells into transplant recipients may provide an approach to prevent cell-mediated rejection of grafts and potentially induce tolerance in the pig to baboon xenotransplantation preclinical model.

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.

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.

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.

BACKGROUND: Xenotransplantation of pig organs may provide an approach to alleviate the severe shortage of human organs. Natural antibodies against Galalpha(1,3)-Gal (alphaGal) epitopes cause hyperacute rejection of pig organs in primates. However, evidence for the role of alphaGal in the natural killer (NK) cell-mediated xenoresponse has been contradictory. METHODS: We investigated the recognition of alphaGal by human NK cells using endo-beta-galactosidase C, an enzyme that cleaves alphaGal, and endothelial cells (EC) from alpha1,3-galactosyltransferase null pigs that do not synthesize alphaGal. Endo-beta-galactosidase C treatment variably reduced the susceptibility of porcine EC to lysis by fresh human NK cells. RESULTS: Removal of alphaGal from porcine EC using endo-beta-galactosidase C, produced variable results, i.e. cytotoxicity was decreased in half of the human NK cell donors tested. The two EC strains from alphaGal-/- pigs were marginally, and not significantly, less susceptible to lysis by naïve human NK cells compared with alphaGal-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)-alpha, 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 alphaGal-/- 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-alpha stimulation, that could explain the differential susceptibility to lysis. Strikingly, all alphaGal-/- 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 alphaGal, and donor variability plays a major role in whether alphaGal has a role in determining susceptibility of porcine EC to lysis. Moreover, susceptibility to lysis of alphaGal null EC is enhanced to the level of wild type EC by activation of either effector or target cells. Elimination of alphaGal alone from source pigs will be insufficient to circumvent the NK cell mediated destruction of porcine EC.

Human natural killer cell activity against porcine targets: modulation by control of the oxidation-reduction environment and role of adhesion molecule interactions.

Xenotransplantation, especially using porcine sources, has been proposed as a means to alleviate the shortage of human organs for transplantation. NK cells appear to be important mediators of the xenogeneic immune responses, including the human anti-pig response. Having previously established the redox regulation of NK cell activity against tumor target cells, we now report that the interaction of human NK cells with porcine target cells is also regulated by redox. Thiol-deprivation strongly diminished the capacity of IL-2-activated human NK cells to kill porcine endothelial cells. This inhibition correlated with reduced proliferation and interferon (IFN)-gamma production by IL-2-activated NK cells. For fresh NK cells, pretreatment with diethyl maleate (DEM), which was used to deplete intracellular thiols, reduced lysis of porcine and human targets. Because many adhesion molecules exhibit interspecies recognition, we further investigated whether changes in expression of adhesion molecules might explain our observations. DEM treatment reduced the expression of CD11b and CD29 on fresh NK cells. Monoclonal antibody blocking studies showed that the combination of mAb to CD11b and CD18 reduced lytic activity against both PAEC as well as K562, although other qualitative differences were observed between the porcine and human target cells. These findings suggest that the oxidative stress-induced downregulation of CD18 may be important in modulating cytotoxic activity of fresh NK cells against PAEC and K562 targets through reduced formation of the CD11b/CD18 heterodimer. Thus, the appropriate manipulation of redox status may provide a means to enhance survival of non-human animal tissues in humans through modulation of adhesion molecule expression/interactions.