NK and T cell repertoire is established early after allogeneic HSCT and is profoundly imprinted by CMV reactivation.

Besides genetic influences, non-genetic factors such as graft-versus-host disease (GvHD) and viral infections have been shown as important shapers of the immune reconstitution and diversification processes after hematopoietic stem cell transplantation (HSCT). However, the differential susceptibility to immune modulation by non-genetic factors is not fully understood. We determined to follow the reconstitution of the T cell receptor (TCR) repertoire through immune-sequencing, of natural killer (NK) cells using a 35-marker spectral flow cytometry panel, and in relation to clinical events. Longitudinal investigation was performed on samples derived from 54 HSCT recipients during the first-year post-HSCT. We confirmed a significant contraction in TCR repertoire diversity with a remarkable stability over time. CMV reactivation had the ability to significantly change TCR repertoire clonality and composition, with a long-lasting imprint. Our data further revealed skewing of NK cell reconstitution in CMV reactivated recipients, with an increased frequency of KIR2DL2L3S2+ adaptive, cytolytic and functional CD107a+ NK cells concomitant with a reduced pool of NKG2A+ NK cells. We provided support that CMV might act as one of the more important driver of peripheral homeostatic proliferation of circulating specific T and NK cells, which can be viewed as a compensatory mechanism to establish a new peripheral repertoire.

Regulation of HLA class I expression by non-coding gene variations.

The Human Leukocyte Antigen (HLA) is a critical genetic system for different outcomes after solid organ and hematopoietic cell transplantation. Its polymorphism is usually determined by molecular technologies at the DNA level. A potential role of HLA allelic expression remains under investigation in the context of the allogenic immune response between donors and recipients. In this study, we quantified the allelic expression of all three HLA class I loci (HLA-A, B and C) by RNA sequencing and conducted an analysis of expression quantitative traits loci (eQTL) to investigate whether HLA expression regulation could be associated with non-coding gene variations. HLA-B alleles exhibited the highest expression levels followed by HLA-C and HLA-A alleles. The max fold expression variation was observed for HLA-C alleles. The expression of HLA class I loci of distinct individuals demonstrated a coordinated and paired expression of both alleles of the same locus. Expression of conserved HLA-A~B~C haplotypes differed in distinct PBMC's suggesting an individual regulated expression of both HLA class I alleles and haplotypes. Cytokines TNFα /IFNß, which induced a very similar upregulation of HLA class I RNA and cell surface expression across alleles did not modify the individually coordinated expression at the three HLA class I loci. By identifying cis eQTLs for the HLA class I genes, we show that the non-coding eQTLs explain 29%, 13%, and 31% of the respective HLA-A, B, C expression variance in unstimulated cells, and 9%, 23%, and 50% of the variance in cytokine-stimulated cells. The eQTLs have significantly higher effect sizes in stimulated cells compared to unstimulated cells for HLA-B and HLA-C genes expression. Our data also suggest that the identified eQTLs are independent from the coding variation which defines HLA alleles and thus may be influential on intra-allele expression variability although they might not represent the causal eQTLs.

Detection of circulating highly expanded T-cell clones in at-risk individuals for rheumatoid arthritis before the clinical onset of the disease.

OBJECTIVES: To quantitatively profile the T-cell repertoire in the peripheral blood of individuals genetically at risk for RA, namely first-degree relatives of RA patients (RA-FDR) at different phases of disease development. METHODS: Next-generation sequencing of the TCR CDR3ß repertoire was performed on genomic DNA isolated from whole blood samples of RA-FDR selected at three different pre-clinical stages and of matched RA patients (n = 20/group). T-cell clones were identified by their unique sequence and their degree of expansion (frequency) within each sample was characterized. Clones with a frequency over 0.5% were considered highly expanded clones (HEC). RESULTS: The absolute number of HEC was significantly higher in established RA patients (mean 4.65) and tended to be higher in symptomatic RA-FDR (mean 3.4) compared with asymptomatic RA-FDR (mean 1.55, P =0.003 and P =0.07, respectively). Asymptomatic individuals with high levels of ACPA did not differ from asymptomatic RA-FDR in terms of absolute number and frequency of clones. The number of HEC tended to be slightly higher at the time of RA onset (P =0.055). Neither clones shared by several patients, nor clones previously associated with RA, were preferentially present within or between the different groups. Finally, a longitudinal analysis did not allow to uncover a kinetic expansion of RA-specific clones closely correlated with disease development. CONCLUSIONS: HEC were detected in the peripheral blood before the clinical onset of RA, in particular in the later pre-clinical phase of RA development, and their presence increased over time.

Natural Killer Cell Alloreactivity Against Human Induced Pluripotent Stem Cells and Their Neuronal Derivatives into Dopaminergic Neurons.

In recent years, great hope has arisen surrounding human stem cells, particularly human induced pluripotent stem (hiPS) cells, as nearly all human tissues can be derived from hiPS cells, using a specific protocol. Therefore, hiPS cells can be a source for replacing defective tissues and make up for the lack of organ donors. However, the alloreactivity of hiPS cells and their derivatives in the context of transplantation remain unclear. Although immunosuppressive drugs can inhibit the T cell compartment, these drugs inhibit partially or not at all natural killer (NK) cells activity. Therefore, the alloreactivity of NK cells against transplanted cells remains to be established. To partially answer this question, we choose, as a model, the potential of cellular therapy for Parkinson's disease (PD). First, we established the in vitro derivation of hiPS cells into mature dopaminergic (mDOPA) neurons, going through an intermediate step called neurosphere (NS) cells. These different cells population were cultured with or without interferon gamma (IFN-γ). They were characterized phenotypically regarding their morphology, and the expression of specific ligands for NK cell receptors expressed by these cells types was investigated. NK cells were isolated from the peripheral blood of healthy donors and cultured in the presence of interleukin 15, to be activated. To test NK cell alloreactivity, a cytotoxic assay was performed with hiPS cells, NS cells, and mDOPA neurons (IFN-γ treated or not) cocultured with allogenic NK cells. Our results show that allogenic NK cells kill hiPS cells (IFN-γ treated or not), but IFN-γ-treated NS cells were protected from killing by allogenic NK cells, compared with untreated NS cells. Finally, mDOPA neurons (IFN-γ treated or not) were partially protected against allogenic NK cell killing. These results indicate that derivatives of hiPS cells, especially NS cells, could be a good product for allogenic transplantation in cellular therapy for PD.

CD8+ T-Cell Repertoire in Human Leukocyte Antigen Class I-Mismatched Alloreactive Immune Response.

In transplantation, direct allorecognition is a complex interplay between T-cell receptors (TCR) and HLA molecules and their bound peptides expressed on antigen-presenting cells. In analogy to HLA mismatched hematopoietic stem cell transplantation (HSCT), the TCR CDR3ß repertoires of alloreactive cytotoxic CD8+ responder T cells, defined by the cell surface expression of CD137 and triggered in vitro by HLA mismatched stimulating cells, were analyzed in different HLA class I mismatched combinations. The same HLA mismatched stimulatory cells induced very different repertoires in distinct but HLA identical responders. Likewise, stimulator cells derived from HLA identical donors activated CD8+ cells expressing very different repertoires in the same mismatched responder. To mimic in vivo inflammation, expression of HLA class l antigens was upregulated in vitro on stimulating cells by the inflammatory cytokines TNFα and IFNß. The repertoires differed whether the same responder cells were stimulated with cells treated or not with both cytokines. In conclusion, the selection and expansion of alloreactive cytotoxic T-cell clonotypes expressing a very diverse repertoire is observed repeatedly despite controlling for HLA disparities and is significantly influenced by the inflammatory status. This makes prediction of alloreactive T-cell repertoires a major challenge in HLA mismatched HSCT.

Niche anchorage and signaling through membrane-bound Kit-ligand/c-kit receptor are kinase independent and imatinib insensitive.

Kit ligand (KitL) and its tyrosine kinase receptor c-kit are critical for germ cells, melanocytes, mastocytes, and hematopoietic stem cells. Alternative splicing of KitL generates membrane-bound KitL (mb-KitL) or soluble KitL, providing survival or cell migration, respectively. Here we analyzed whether c-kit can function both as an adhesion and signaling receptor to mb-KitL presented by the environmental niche. At contacts between fibroblasts and MC/9 mast cells, mb-KitL, and c-kit formed ligand/receptor clusters that formed stable complexes, which resisted dissociation by c-kit blocking mAbs and provided cell anchorage under physiological shear stresses. Clusters recruited tyrosine-phosphorylated proteins and induced spatially restricted F-actin polymerization. Mutational analysis of c-kit demonstrated kinase-independent mb-KitL/c-kit clustering, anchorage, F-actin polymerization, and Tyr567-dependent cluster phosphorylation. Kinase inhibition of c-kit by imatinib reduced cluster coalescence, but allowed cluster phosphorylation and F-actin polymerization, which required PI3K recruitment and a newly identified juxtamembrane residue. Synergies between integrin and c-kit-mediated spreading and adhesion of MC/9 cells were studied in vitro on immobilized-KitL/fibronectin surfaces. While c-kit blocking antibodies prevented spreading, imatinib blocked spreading induced by soluble- but not immobilized KitL. Thus, "mechanical" activation of c-kit provides signaling, niche-anchorage, and synergies with integrin-mediated adhesion, which is independent of kinase function and resistant to c-kit kinase inhibitors.-