Novel Approach to Cell Surface Discrimination Between KIR2DL1 Subtypes and KIR2DS1 Identifies Hierarchies in NK Repertoire, Education, and Tolerance

Cumulative activating and inhibitory receptor signaling controls the functional output of individual natural killer (NK) cells. Investigation of how competing signals impact response, however, has been hampered by the lack of available antibodies capable of distinguishing inhibitory and activating receptors with highly homologous ectodomains. Utilizing a novel combination of monoclonal antibodies with specificity for discrete inhibitory KIR2DL1 and activating KIR2DS1 allotypes found among 230 healthy donors, we investigated allele-specific receptor expression and function driven by KIR2DL1 and KIR2DS1 alleles. We found that co-expression of the HLA-C2 ligand diminishes KIR2DL1, but not KIR2DS1, cell surface staining, but does not impact the respective frequencies of KIR2DL1- and KIR2DS1-expressing cells within the NK repertoire. We can distinguish by flow cytometry NK cell populations expressing the most common KIR2DL1-C245 allotypes from those expressing the most common KIR2DL1-R245 allotypes, and we show that the informative differential binding anti-KIR2DL1/S1 clone 1127B is determined by amino acid residue T154. Although both KIR2DL1-C245 and KIR2DL1-R245 subtypes can be co-expressed in the same cell, NK cells preferentially express one or the other. Cells expressing KIR2DL1-C245 exhibited a lower KIR2DL1 cell surface density and lower missing-self reactivity in comparison to cells expressing KIR2DL1-R245. We found no difference, however, in sensitivity to inhibition or cell surface stability between the two KIR2DL1 isoforms, and both demonstrated similar expansion among NKG2C+ KIR2DL1+ NK cells in HCMV-seropositive healthy individuals. In addition to cell surface density of KIR2DL1, copy number of cognate HLA-C2 hierarchically impacted the effector capacity of both KIR2DL1+ cells and the tolerization of KIR2DS1+ NK cells. HLA-C2 tolerization of KIR2DS1+ NK cells could be overridden, however, by education via co-expressed self-specific inhibitory receptors, such as the heterodimer CD94/NKG2A. Our results demonstrate that effector function of NK cells expressing KIR2DL1 or KIR2DS1 is highly influenced by genetic variability and is calibrated by co-expression of additional NK receptors and cognate HLA-C2 ligands. These findings define the molecular conditions under which NK cells are activated or inhibited, potentially informing selection of donors for adoptive NK therapies

iNKT cell development is orchestrated by different branches of TGF-β signaling

Invariant natural killer T (iNKT) cells constitute a distinct subset of T lymphocytes exhibiting important immune-regulatory functions. Although various steps of their differentiation have been well characterized, the factors controlling their development remain poorly documented. Here, we show that TGF-β controls the differentiation program of iNKT cells. We demonstrate that TGF-β signaling carefully and specifically orchestrates several steps of iNKT cell development. In vivo, this multifaceted role of TGF-β involves the concerted action of different pathways of TGF-β signaling. Whereas the Tif-1γ branch controls lineage expansion, the Smad4 branch maintains the maturation stage that is initially repressed by a Tif-1γ/Smad4-independent branch. Thus, these three different branches of TGF-β signaling function in concert as complementary effectors, allowing TGF-β to fine tune the iNKT cell differentiation program

Recherche et étude de gènes différentiellement exprimés dans des modèles de tolérance à l'allogreffe chez le rat

Par des méthodes de recherche de gènes différentiellement exprimés, nous avons identifié deux molécules jouant potentiellement un rôle dans les mécanismes de tolérance. Dans un premier modèle basé sur l'administration d'un immunosuppresseur, le LF15-0195, nous avons identifié Schlafen-3 (Slfn3). surexprimé dans les LT d'animaux tolérants. Nous montrons que Slfn3 est un marqueur non exclusif des cellules T naturelles régulatrices CD4+CD25 mais également un marqueur de l'activation des cellules T. Dans un deuxième modèle basé sur l'induction de tolérance par Transfusion Sanguine du Donneur (TSD), nous avons identifié le gène codant pour la molécule Follistatin-Like 1 (FSTL1) surexprimée dans les allogreffes tolérées. Nous montrons que FSTL1 est une molécule exprimée par les cellules T CD8. infiltrant la greffe. La surexpression de FSTL1 à l'aide de vecteurs viraux permet de prolonger significativement la survie de l'allogreffe, suggérant un rôle immunomodulateur de cette molécule.Using genes searching methods, we identified two molecules playing a potential role in the tolerance mechanisms. In a first model, based on the administration of an immunosuppressor, LF15-0195, we identified of Schlafen-3 (Slfn3), which is overexpressed in LT of tolerant animals. We showed that Slfn3 is a non exclusive marker of CD4+CD25+ natural regulatory T cells, but is also a marker of T cell activation. In a second model, based on the induction of tolerance by Donor Specific Transfusion (DST), we identified a gene coding for the Follistatin-Like 1 molecule (FSTL1), which is overexpressed in tolerated allografts. We showed that FSTL1 is expressed by T CD8+ graft infiltrating cells. The FSTL1 overexpression using viral vectors have a prolongation effect on allograft survival, suggesting an immunoregulator role of this molecule.NANTES-BU Médecine pharmacie (441092101) / SudocSudocFranceF

Novel multiplex PCR-SSP method for centromeric KIR allele discrimination

Abstract Allelic diversity of the KIR2DL receptors drive differential expression and ligand-binding affinities that impact natural killer cell function and patient outcomes for diverse cancers. We have developed a global intermediate resolution amplification-refractory mutation system (ARMS) PCR-SSP method for distinguishing functionally relevant subgroups of the KIR2DL receptors, as defined by phylogenetic study of the protein sequences. Use of the ARMS design makes the method reliable and usable as a kit, with all reactions utilizing the same conditions. Six reactions define six subgroups of KIR2DL1; four reactions define three subgroups of KIR2DL2; and five reactions define four subgroups of KIR2DL3. Using KIR allele data from a cohort of 426 European-Americans, we identified the most common KIR2DL subtypes and developed the high-throughput PCR-based methodology, which was validated on a separate cohort of 260 healthy donors. Linkage disequilibrium analysis between the different KIR2DL alleles revealed that seven allelic combinations represent more than 95% of the observed population genotypes for KIR2DL1/L2/L3. In summary, our findings enable rapid typing of the most common KIR2DL receptor subtypes, allowing more accurate prediction of co-inheritance and providing a useful tool for the discrimination of observed differences in surface expression and effector function among NK cells exhibiting disparate KIR2DL allotypes

PCR reaction conditions.

<p>PCR reaction conditions.</p

Distribution of KIR3DL1 alleles and primer target sites.

<p>Distribution of KIR3DL1 alleles and primer target sites.</p

PCR primers and reaction conditions.

a<p>internal control primers and ConsF have been described previously (2, 23).</p

Development of a Novel Multiplex PCR Assay to Detect Functional Subtypes of KIR3DL1 Alleles

<div><p>Among NK cell receptor-ligand partnerships, KIR3DL1 and HLA-Bw4 demonstrate the greatest diversity; permutations of their allelic combinations titrate NK reactivity. Balancing selection has maintained distinct subtypes of KIR3DL1 alleles in global populations, implying that each may provide unique fitness advantages and variably influence disease processes. Though approaches exist for determining HLA-B allotypes, practical methods for identifying KIR3DL1 alleles are lacking. We have developed a PCR-based approach that identifies functional subtypes of KIR3DL1 alleles; it is suitable for research and may have clinical application. Six allele subsets were identified based on expression characteristics of the eleven most common KIR3DL1 alleles represented in reported populations. The remaining 62 low-frequency alleles were distributed into these groups based on sequence homology to coding regions. Subtype-specific SNPs were found in exons 3, 4, and 7, and used as priming sites for five multiplex PCR reactions. Genomic DNA derived from 175 unrelated donors and 52 related individuals from 6 families demonstrated >99.5% concordance between sequence-based typing and our novel approach. Finally, PCR-based typing accurately predicted NK phenotypes obtained by flow cytometry after staining with DX9 and Z27 monoclonal antibodies. This novel approach facilitates high-throughput analysis of KIR3DL1 allotypes to enable a broader understanding of KIR3DL1 and HLA-Bw4 interaction in health and disease.</p></div

Multiplex PCR assessment accurately predicts KIR3DL1 allotypes.

<p>(A) KIR3DL1 subtyping was completed for three CEPH parent-child quartets and matched previous KIR3DL1 allele typing <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099543#pone.0099543-Halfpenny1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099543#pone.0099543-Sun1" target="_blank">[27]</a>. Paternal alleles are shown in blue; maternal in red. (B) KIR3DL1 subtype analysis of three generations of CEPH family individuals demonstrates Mendelian inheritance. Non-inherited alleles present in grandparents are indicated by grey shading. (C) Analysis of CEPH family 1416 demonstrates inheritance of a polyallelic haplotype, encoding both KIR3DS1 and a KIR3DL1-null allele.</p

KIR3DL1 subgroup typing accurately predicts NK phenotypes by multi-parameter flow cytometry.

<p>NK cells from eight representative healthy donors were surface-stained with DX9 and Z27 antibodies. KIR3DL1 subtypes identified by the multiplex typing method have known expression characteristics and they are indicated below each of the eight flow cytometry plots shown. KIR3DL1 subtyping and NK phenotypes were consistent for all 104 individuals tested.</p