Grandmaternal cells in cord blood.

BACKGROUND: During pregnancy a feto-maternal exchange of cells through the placenta conducts to maternal microchimerism (Mc) in the child and fetal Mc in the mother. Because of this bidirectional traffic of cells, pregnant women have also acquired maternal cells in utero from their mother and could transfer grandmaternal (GdM) cells to their child through the maternal bloodstream during pregnancy. Thus, cord blood (CB) samples could theoretically carry GdMMc. Nevertheless this has never been demonstrated. METHODS: Using Human Leukocyte Antigen (HLA)-specific quantitative PCR assays on three-generation families, we were able to test 28 CB samples from healthy primigravid women for GdMMc in whole blood (WB) and isolated cells (PBMC, T, B, granulocytes, stem cells). FINDINGS: Five CB samples (18%) had GdMMc which could not be confounded with maternal source, with quantities 100 fold lower than maternal Mc in WB and PBMC. Risk of aneuploidies and/or related invasive prenatal procedures significantly correlated with the presence of GdMMc in CB (p=0.024). Significantly decreased HLA compatibility was observed in three-generation families from CB samples carrying GdMMc (p=0.019). INTERPRETATION: Transgenerational transfer of cells could have implications in immunology and evolution. Further analyses will be necessary to evaluate whether GdMMc in CB is a passive or immunologically active transfer and whether invasive prenatal procedures could trigger GdMMc. FUNDING: Provence-Alpes-Côte d'Azur APEX grant # 2012_06549E, 2012_11786F and 2014_03978) and the Foundation for Medical Research (FRM Grant #ING20140129045).

Factors Predicting the Presence of Maternal Cells in Cord Blood and Associated Changes in Immune Cell Composition.

Background: Cord blood (CB) samples are increasingly used as a source of hematopoietic stem cells in transplantation settings. Maternal cells have been detected in CB samples and their presence is associated with a better graft outcome. However, we still do not know what influences the presence of maternal microchimerism (MMc) in CB samples and whether their presence influences CB hematopoietic cell composition. Patients and Methods: Here we test whether genetic, biological, anthropometric and/or obstetrical parameters influence the frequency and/or quantity of maternal Mc in CB samples from 55 healthy primigravid women. Mc was evaluated by targeting non-shared, non-inherited Human Leukocyte Antigen (HLA)-specific real-time quantitative PCR in whole blood and four cell subsets (T, B lymphocytes, granulocytes and/or hematopoietic progenitor cells). Furthermore CB samples were analyzed for their cell composition by flow cytometry and categorized according to their microchimeric status. Results: MMc was present in 55% of CB samples in at least one cell subset or whole blood, with levels reaching up to 0.3% of hematopoietic progenitor cells. Two factors were predictive of the presence of MMc in CB samples: high concentrations of maternal serological Pregnancy-Associated-Protein-A at first trimester of pregnancy (p=0.018) and feto-maternal HLA-A and/or -DR compatibility (p=0.009 and p=0.01 respectively). Finally, CB samples positive for MMc were significantly enriched in CD56+ cells compared to CB negative for MMc. Conclusions: We have identified two factors, measurable at early pregnancy, predicting the presence of maternal cells in CB samples at delivery. We have shown that MMc in CB samples could have an influence on the hematopoietic composition of fetal cells. CD56 is the phenotypic marker of natural killer cells (NK) and NK cells are known to be the main effector for graft versus leukemia reactions early after hematopoietic stem cell transplantation. These results emphasize the importance of MMc investigation for CB banking strategies.

Mosaicism of XX and XXY cells accounts for high copy number of Toll like Receptor 7 and 8 genes in peripheral blood of men with Rheumatoid Arthritis.

The X chromosome, hemizygous in males, contains numerous genes important to immunological and hormonal function. Alterations in X-linked gene dosage are suspected to contribute to female predominance in autoimmunity. A powerful example of X-linked dosage involvement comes from the BXSB murine lupus model, where the duplication of the X-linked Toll-Like Receptor 7 (Tlr7) gene aggravates autoimmunity in male mice. Such alterations are possible in men with autoimmune diseases. Here we showed that a quarter to a third of men with rheumatoid arthritis (RA) had significantly increased copy numbers (CN) of TLR7 gene and its paralog TLR8. Patients with high CN had an upregulated pro-inflammatory JNK/p38 signaling pathway. By fluorescence in situ hybridization, we further demonstrated that the increase in X-linked genes CN was due to the presence of an extra X chromosome in some cells. Men with RA had a significant cellular mosaicism of female (46,XX) and/or Klinefelter (47,XXY) cells among male (46,XY) cells, reaching up to 1.4% in peripheral blood. Our results present a new potential trigger for RA in men and opens a new field of investigation particularly relevant for gender-biased autoimmune diseases.

Evaluation of X Chromosome Inactivation with Respect to HLA Genetic Susceptibility in Rheumatoid Arthritis and Systemic Sclerosis.

BACKGROUND: Autoimmune diseases, including rheumatoid arthritis (RA) and systemic sclerosis (SSc) are characterized by a strong genetic susceptibility from the Human Leucocyte Antigen (HLA) locus. Additionally, disorders of epigenetic processes, in particular non-random X chromosome inactivation (XCI), have been reported in many female-predominant autoimmune diseases. Here we test the hypothesis that women with RA or SSc who are strongly genetically predisposed are less susceptible to XCI bias. METHODS: Using methylation sensitive genotyping of the androgen receptor (AR) gene, XCI profiles were performed in peripheral blood mononuclear cells from 161 women with RA, 96 women with SSc and 100 healthy women. HLA-DRB1 and DQB1 were genotyped. Presence of specific autoantibodies was documented for patients. XCI skewing was defined as having a ratio ≥ 80:20 of cells inactivating the same X chromosome. RESULTS: 110 women with RA, 68 women with SSc, and 69 controls were informative for the AR polymorphism. Among them 40.9% of RA patients and 36.8% of SSc patients had skewed XCI compared to 17.4% of healthy women (P = 0.002 and 0.018, respectively). Presence of RA-susceptibility alleles coding for the "shared epitope" correlated with higher skewing among RA patients (P = 0.002) and such correlation was not observed in other women, healthy or with SSc. Presence of SSc-susceptibility alleles did not correlate with XCI patterns among SSc patients. CONCLUSION: Data demonstrate XCI skewing in both RA and SSc compared to healthy women. Unexpectedly, skewed XCI occurs more often in women with RA carrying the shared epitope, which usually reflects severe disease. This reinforces the view that loss of mosaicism in peripheral blood may be a consequence of chronic autoimmunity.

Anti-Ephrin Type-B Receptor 2 (EphB2) and Anti-Three Prime Histone mRNA EXonuclease 1 (THEX1) Autoantibodies in Scleroderma and Lupus.

In a pilot ProtoArray analysis, we identified 6 proteins out of 9483 recognized by autoantibodies (AAb) from patients with systemic sclerosis (SSc). We further investigated the 6 candidates by ELISA on hundreds of controls and patients, including patients with Systemic Lupus Erythematosus (SLE), known for high sera reactivity and overlapping AAb with SSc. Only 2 of the 6 candidates, Ephrin type-B receptor 2 (EphB2) and Three prime Histone mRNA EXonuclease 1 (THEX1), remained significantly recognized by sera samples from SSc compared to controls (healthy or with rheumatic diseases) with, respectively, 34% versus 14% (P = 2.10-4) and 60% versus 28% (P = 3.10-8). Above all, EphB2 and THEX1 revealed to be mainly recognized by SLE sera samples with respectively 56%, (P = 2.10-10) and 82% (P = 5.10-13). As anti-EphB2 and anti-THEX1 AAb were found in both diseases, an epitope mapping was realized on each protein to refine SSc and SLE diagnosis. A 15-mer peptide from EphB2 allowed to identify 35% of SLE sera samples (N = 48) versus only 5% of any other sera samples (N = 157), including SSc sera samples. AAb titers were significantly higher in SLE sera (P<0.0001) and correlated with disease activity (p<0.02). We could not find an epitope on EphB2 protein for SSc neither on THEX1 for SSc or SLE. We showed that patients with SSc or SLE have AAb against EphB2, a protein involved in angiogenesis, and THEX1, a 3'-5' exoribonuclease involved in histone mRNA degradation. We have further identified a peptide from EphB2 as a specific and sensitive tool for SLE diagnosis.