HLA-Cw4 expression on porcine endothelial cells reduces cytotoxicity and adhesion mediated by CD158a+ human NK cells.

BACKGROUND: Human natural killer (NK) cell-mediated cytotoxicity represents a hurdle in pig-to-human xenotransplantation. It was previously reported that the expression of human major histocompatibility complex class I molecules, including HLA-B27, -Cw3, -E, and -G, partially protects porcine endothelial cells (pEC) from human NK-mediated cytotoxicity and that HLA-G inhibits NK adhesion to pEC. Here, we tested if HLA-Cw4 expression on pEC alone, or concurrently with HLA-Cw3, prevents human NK adhesion and cytotoxicity against pEC via recognition of the killer-cell immunoglobulin-like receptors (KIR) CD158a (KIR2DL1) and CD158b (KIR2DL2/3), respectively. METHODS: Two pEC lines (2A2 and PEDSV.15) were transfected with HLA-Cw3 and HLA-Cw4. HLA and KIR expression on porcine and human cells were analyzed by flow cytometry. The effect of HLA expression on pEC on human NK-mediated cytotoxicity and adhesion was tested by (51)Cr-release and dynamic adhesion assays, respectively. RESULTS: HLA-Cw4 expression on pEC reduced cytotoxicity mediated by CD158a(+) polyclonal human NK cells by an average of 58%, and by CD158a(bright) NK cell clones by 68%, but not by NK cells expressing low levels of CD158. Co-expression of HLA-Cw3 and HLA-Cw4 on pEC did not mediate further protection against NK cytotoxicity. The expression of HLA-Cw4 reduced the adhesion of human NK cells on pEC by a mean of 53%. CONCLUSIONS: While transgenic expression of HLA-Cw4 on pEC reduces NK cell adhesion and cytotoxicity, co-expression with HLA-Cw3 is not sufficient to completely overcome human NK-mediated cytotoxicity in vitro.

Functional activity of maternal and cord antibodies elicited by an investigational group B Streptococcus trivalent glycoconjugate vaccine in pregnant women.

OBJECTIVES: The main aim of this exploratory study was to evaluate functional activity of antibodies elicited by a maternal Group B Streptococcus (GBS) investigational vaccine composed of capsular polysaccharides Ia, Ib, and III conjugated to genetically detoxified Diphtheria toxin CRM197. The second objective was to investigate the relationship between serotype-specific IgG concentrations and functional activity in maternal and cord sera. METHODS: Maternal and cord sera collected at baseline and at delivery from vaccine and placebo recipients during a double-blind placebo-controlled Phase II study (www.clinicaltrials.gov, NCT01446289) were tested in an opsono-phagocytic bacterial killing assay. Cord sera from vaccine recipients were also passively transferred to newborn mice to investigate conferred protection against bacterial challenge. RESULTS: Antibody-mediated GBS phagocytic killing was significantly increased in maternal serum at delivery and in cord sera from the investigational vaccine group as compared to the placebo group. Anti-capsular IgG concentrations above 1 µg/mL mediated in vitro killing against GBS strains belonging to all three serotypes and IgG levels correlated with functional titers. Passively administered cord sera elicited a dose-dependent protective response against all GBS serotypes in the in vivo model. CONCLUSIONS: The maternal vaccine elicited functional antibodies that were placentally transferred. Anti-capsular IgG concentrations in maternal and cord sera were predictive of functional activity and in vivo protection in the mouse model.

Maternal Immunization With an Investigational Trivalent Group B Streptococcal Vaccine: A Randomized Controlled Trial.

OBJECTIVE: To evaluate the safety and immunogenicity of an investigational trivalent group B streptococcal vaccine in pregnant women and antibody transfer to their newborns. METHODS: The primary outcome of this observer-blind, randomized study was to estimate placental antibody transfer rates at birth. Secondary outcomes included measurement of serotype-specific antibodies at screening, 30 days postvaccination, at delivery, and 91 days postpartum, infant antibody levels at 3 months of age, the potential effect on routine infant diphtheria vaccination at 1 month after the third infant series dose, and safety in mother and infant participants through at least 5 months postpartum. Sample size was based on 60 participants in the vaccine group giving a probability of observing at least one adverse event of 90% if the actual rate of the event was 3.8%. RESULTS: From September 2011 to October 2013, 86 pregnant women were allocated in a 3:2 ratio to receive an investigational group B streptococcal vaccine containing glycoconjugates of serotypes Ia, Ib, and III or placebo. Demographics were similar across groups. Transfer ratios were 66-79% and maternal geometric mean concentrations increased 16-, 23-, and 20-fold by delivery against serotypes Ia, Ib, and III, respectively, Women with no detectable antibodies at inclusion had lower responses than those with detectable antibodies. Three months after birth, infant antibody concentrations were 22-25% of birth levels. Antidiphtheria geometric mean concentrations were similar across groups. In the vaccine and placebo groups, 32 of 51 women (63%) and 26 of 35 women (74%) reported adverse effects, respectively. CONCLUSION: The investigational vaccine was well-tolerated without safety signals in recipients and their infants or interference with routine infant diphtheria vaccination, although further studies on safety and effectiveness are needed. The investigational vaccine was immunogenic for all serotypes, particularly among women with detectable antibody levels at baseline. Antibody transfer to neonates was at similar levels to other maternally administered polysaccharide vaccines. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, www.clinicaltrials.gov, NCT01446289.

Human CD26 expression in transgenic mice affects murine T-cell populations and modifies their subset distribution.

CD26 is a type II transmembrane glycoprotein with dipeptidyl peptidase (DPPIV) activity, constitutively expressed in different cell types and contributing to T-cell activation by acting as costimulatory molecule. Although data suggest an important role for CD26 within the immune system, the physiologic function of this molecule is still unknown. To investigate the role of CD26 in vivo we have produced transgenic mice expressing the human molecule in T cells. Human CD26 (huCD26) is constitutively expressed in all thymocytes and peripheral T lymphocytes of these transgenic mice and is endowed with an enhanced DPPIV activity. CD26 transgene expression induces major phenotypic changes to T-cell populations within the thymus and in peripheral blood. After the onset of sexual maturity, huCD26 expression induces an age-related overreduction of thymus cellularity accompanied by a relative impairment of thymocyte proliferation following lectin stimulation. Also the peripheral blood T-cell pool is reduced in huCD26 transgenic mice and this is accompanied by an increase of the apoptotic rate of CD4+ and CD8+ subpopulations. Taken together these data suggest that CD26 interferes with transduction pathway(s) needed for the maturation of T cells and plays an important role in T lymphocyte homeostasis in peripheral blood.

Transgenic expression of HLA-E single chain trimer protects porcine endothelial cells against human natural killer cell-mediated cytotoxicity.

BACKGROUND: The susceptibility of porcine endothelial cells (pEC) to human natural killer (NK) cells is related to the failure of human major histocompatibility complex (MHC)-specific killer inhibitory receptors to recognize porcine MHC class I molecules. The aims of this study were (i) to assess the protection of pEC against xenogeneic NK-mediated cytotoxicity afforded by the stable expression of HLA-E single chain trimers (SCT) composed of a canonical HLA-E binding peptide antigen, VMAPRTLIL, the mature human beta2-microglobulin, and the mature HLA-E heavy chain, and (ii) to test whether HLA-E expression on pEC and porcine lymphoblastoid cells affects the adhesion of human NK cells. METHODS: Porcine EC lines expressing different levels of HLA-E SCT were generated by Ca(2)PO(4)-transfection followed by limiting dilution cloning. Surface expression of HLA-E was measured by flow cytometry. Susceptibility of transfected pEC lines against human NK cells was tested in (51)Cr-release cytotoxicity assays. Interactions between human NK cells and HLA-E positive pEC or porcine lymphoblastoid cells were further addressed in adhesion and conjugation assays. RESULTS: The level of protection of pEC from human NK-mediated cytotoxicity correlated with the intensity of surface HLA-E expression. Furthermore, the HLA-E SCT-mediated protection was specifically reversed by blocking the HLA-E specific NK inhibitory receptor CD94/NKG2A. HLA-E expression does neither affect the adhesion of human NK cells to pEC nor the heteroconjugate formation between human NK and porcine 13271.10 cells. CONCLUSIONS: Stable surface expression of HLA-E on pEC was achieved in the absence of extrinsic peptide pulsing and provided partial protection from human NK cytotoxicity. Though insufficient to inhibit xenogeneic NK cell reactivity completely, transgenic HLA-E expression on pig organs might contribute to a successful application of clinical xenotransplantation in combination with other protective strategies.

HLA-E expression on porcine cells: protection from human NK cytotoxicity depends on peptide loading.

Human NK cells lyse porcine cells and may play an important role in the cell-mediated rejection of pig-to-human xenografts. Lysis is probably a consequence of the failure of human MHC-specific killer inhibitory receptors to recognize porcine MHC class I molecules. A majority of activated human NK cells express the HLA-E-specific inhibitory receptor CD94/NKG2A. The aim of this study was therefore to test the hypothesis that stable surface expression of HLA-E on porcine cells protects against xenogeneic NK-mediated cytotoxicity. Porcine lymphoblastoid (13 271) and endothelial (pEC) cell lines were transfected with constructs coding for HLA-E together with the leader sequence of HLA-B7 or -A2. HLA-E was correctly expressed on 13 271 cells while pEC required peptide-pulsing and/or IFN-gamma stimulation to express the HLA-E complex on the cell surface. HLA-E-expressing porcine cells were partially protected from lysis mediated by human polyclonal NK populations and completely protected from killing by NKG2Abright NK clones. In conclusion, the capability of different porcine cell types to express HLA-E on the cell surface can differ considerably depending decisively on the availability of peptides. These findings are important for the applicability of transgenic HLA-E expression as an approach to protect porcine tissues from human NK cytotoxicity.

Human NK cytotoxicity against porcine cells is triggered by NKp44 and NKG2D.

Pig-to-human xenotransplantation has been proposed as a means to alleviate the shortage of human organs for transplantation, but cellular rejection remains a hurdle for successful xenograft survival. NK cells have been implicated in xenograft rejection and are tightly regulated by activating and inhibitory receptors recognizing ligands on potential target cells. The aim of the present study was to analyze the role of activating NK receptors including NKp30, NKp44, NKp46, and NKG2D in human xenogeneic NK cytotoxicity against porcine endothelial cells (pEC). (51)Cr release and Ab blocking assays were performed using freshly isolated, IL-2-activated polyclonal NK cell populations as well as a panel of NK clones. Freshly isolated NK cells are NKp44 negative and lysed pEC exclusively in an NKG2D-dependent fashion. In contrast, the lysis of pEC mediated by activated human NK cells depended on both NKp44 and NKG2D, since a complete protection of pEC was achieved only by simultaneous blocking of these activating NK receptors. Using a panel of NK clones, a highly significant correlation between anti-pig NK cytotoxicity and NKp44 expression levels was revealed. Other triggering receptors such as NKp30 and NKp46 were not involved in xenogeneic NK cytotoxicity. Finally, Ab-dependent cell-mediated cytotoxicity of pEC mediated by human NK cells in the presence of xenoreactive Ab was not affected by blocking of activating NK receptors. In conclusion, strategies aimed to inhibit interactions between NKp44 and NKG2D on human NK cells and so far unknown ligands on pEC may prevent direct NK responses against xenografts but not xenogeneic Ab-dependent cell-mediated cytotoxicity.

Lack of galactose-alpha-1,3-galactose expression on porcine endothelial cells prevents complement-induced lysis but not direct xenogeneic NK cytotoxicity.

The galactose-alpha-1,3-galactose (alphaGal) carbohydrate epitope is expressed on porcine, but not human cells, and therefore represents a major target for preformed human anti-pig natural Abs (NAb). Based on results from pig-to-primate animal models, NAb binding to porcine endothelial cells will likely induce complement activation, lysis, and hyperacute rejection in pig-to-human xenotransplantation. Human NK cells may also contribute to innate immune responses against xenografts, either by direct recognition of activating molecules on target cells or by FcgammaRIII-mediated xenogeneic Ab-dependent cellular cytotoxicity (ADCC). The present study addressed the question as to whether the lack of alphaGal protects porcine endothelial cells from NAb/complement-induced lysis, direct xenogeneic NK lysis, NAb-dependent ADCC, and adhesion of human NK cells under shear stress. Homologous recombination, panning, and limiting dilution cloning were used to generate an alphaGal-negative porcine endothelial cell line, PED2*3.51. NAb/complement-induced xenogeneic lysis of PED2*3.51 was reduced by an average of 86% compared with the alphaGal-positive phenotype. PED2*3.51 resisted NK cell-mediated ADCC with a reduction of lysis ranging from 30 to 70%. However, direct xenogeneic lysis of PED2*3.51, mediated either by freshly isolated or IL-2-activated human NK cells or the NK cell line NK92, was not reduced. Furthermore, adhesion of IL-2-activated human NK cells did not rely on alphaGal expression. In conclusion, removal of alphaGal leads to a clear reduction in complement-induced lysis and ADCC, but does not resolve adhesion of NK cells and direct anti-porcine NK cytotoxicity, indicating that alphaGal is not a dominant target for direct human NK cytotoxicity against porcine cells.

Xenogeneic human NK cytotoxicity against porcine endothelial cells is perforin/granzyme B dependent and not inhibited by Bcl-2 overexpression.

Because of organ shortages in clinical allotransplantation, the potential of pig-to-human xenotransplantation is currently being explored showing a possible critical role for natural killer (NK) cells in the immune response against xenografts. Therefore, we analyzed the cytotoxic pathways utilized by human natural killer cells (hNK) against porcine endothelial cells (pEC). Transmission electron microscopy of pEC cocultured with hNK cells showed both apoptotic and necrotic cell death, whereas soluble factors such as Fas ligand or TNFalpha did not induce apoptosis in pEC. NK lysis of pEC was abrogated by concanamycin A and ammonium chloride, reagents inhibiting the perforin/granzyme B (grB) pathway, but only partially blocked by caspase inhibition with z-VAD-fmk. Overexpression of bcl-2 protected pEC against apoptosis induced by staurosporine or actinomycin D, but failed to prevent hNK cell-mediated lysis. In conclusion, pEC are lysed in vitro by hNK cells via the perforin/grB pathway and are not protected from NK lysis by overexpression of bcl-2.