Leukocytes with chromosome Y loss have reduced abundance of the cell surface immunoprotein CD99.

Mosaic loss of chromosome Y (LOY) in immune cells is a male-specific mutation associated with increased risk for morbidity and mortality. The CD99 gene, positioned in the pseudoautosomal regions of chromosomes X and Y, encodes a cell surface protein essential for several key properties of leukocytes and immune system functions. Here we used CITE-seq for simultaneous quantification of CD99 derived mRNA and cell surface CD99 protein abundance in relation to LOY in single cells. The abundance of CD99 molecules was lower on the surfaces of LOY cells compared with cells without this aneuploidy in all six types of leukocytes studied, while the abundance of CD proteins encoded by genes located on autosomal chromosomes were independent from LOY. These results connect LOY in single cells with immune related cellular properties at the protein level, providing mechanistic insight regarding disease vulnerability in men affected with mosaic chromosome Y loss in blood leukocytes.

SeXY chromosomes and the immune system: reflections after a comparative study.

BACKGROUND: Sex bias in immune function has been contributed in part to a preponderance of immune system-related genes (ISRG) on the X-chromosome. We verified whether ISRG are more abundant on the X chromosome as compared to autosomal chromosomes and reflected on the impact of our findings. METHODS: Consulting freely accessible databases, we performed a comparative study consisting of three complementary strategies. First, among coding X/Y-linked genes, the abundance of ISRG was compared to the abundance of genes dedicated to other systems. Genes were assigned considering three criteria: disease, tissue expression, and function (DEF approach). In addition, we carried out two genome-wide approaches to compare the contribution of sex and autosomal chromosomes to immune genes defined by an elevated expression in lymphatic tissues (LTEEG approach) or annotation to an immune system process, GO:0002376 (GO approach). RESULTS: The X chromosome had less immune genes than the median of the autosomal chromosomes. Among X-linked genes, ISRG ranked fourth after the reproductive and nervous systems and genes dedicated to development, proliferation and apoptosis. On the Y chromosome, ISRG ranked second, and at the pseudoautosomal region (PAR) first. According to studies on the expression of X-linked genes in a variety of (mostly non-lymphatic) tissues, almost two-thirds of ISRG are expressed without sex bias, and the remaining ISRG presented female and male bias with similar frequency. Various epigenetic controllers, X-linked MSL3 and Y-linked KDM5D and UTY, were preferentially expressed in leukocytes and deserve further attention for a possible role in sex biased expression or its neutralisation. CONCLUSIONS: The X chromosome is not enriched for ISRG, though particular X-linked genes may be responsible for sex differences in certain immune responses. So far, there is insufficient information on sex-biased expression of X/Y-linked ISRG in leukocytes to draw general conclusions on the impact of X/Y-linked ISRG in immune function. More research on the regulation of the expression X-linked genes is required with attention to 1) female and male mechanisms that may either augment or diminish sex biased expression and 2) tissue-specific expression studies.

Maternal microchimerism: increased in the insulin positive compartment of type 1 diabetes pancreas but not in infiltrating immune cells or replicating islet cells.

BACKGROUND: Maternal microchimeric cells (MMc) transfer across the placenta during pregnancy. Increased levels of MMc have been observed in several autoimmune diseases including type 1 diabetes but their role is unknown. It has been suggested that MMc are 1) effector cells of the immune response, 2) targets of the autoimmune response or 3) play a role in tissue repair. The aim of this study was to define the cellular phenotype of MMc in control (n = 14) and type 1 diabetes pancreas (n = 8). METHODS: Using sex chromosome-based fluorescence in-situ hybridization, MMc were identified in male pancreas and their phenotype determined by concomitant immunofluorescence. RESULTS: In normal pancreas, MMc positive for endocrine, exocrine, duct and acinar markers were identified suggesting that these cells are derived from maternal progenitors. Increased frequencies of MMc were observed in type 1 diabetes pancreas (p = 0.03) with particular enrichment in the insulin positive fraction (p = 0.01). MMc did not contribute to infiltrating immune cells or Ki67+ islet cell populations in type 1 diabetes. CONCLUSION: These studies provide support for the hypothesis that MMc in human pancreas are derived from pancreatic precursors. Increased frequencies of MMc beta cells may contribute to the initiation of autoimmunity or to tissue repair but do not infiltrate islets in type 1 diabetes.

HLA-targeted flow cytometric sorting of blood cells allows separation of pure and viable microchimeric cell populations.

Microchimerism is defined by the presence of low levels of nonhost cells in a person. We developed a reliable method for separating viable microchimeric cells from the host environment. For flow cytometric cell sorting, HLA antigens were targeted with human monoclonal HLA antibodies (mAbs). Optimal separation of microchimeric cells (present at a proportion as low as 0.01% in artificial mixtures) was obtained with 2 different HLA mAbs, one targeting the chimeric cells and the other the background cells. To verify purity of separated cell populations, flow-sorted fractions of 1000 cells were processed for DNA analysis by HLA-allele-specific and Y-chromosome-directed real-time quantitative PCR assays. After sorting, PCR signals of chimeric DNA markers in the positive fractions were significantly enhanced compared with those in the presort samples, and they were similar to those in 100% chimeric control samples. Next, we demonstrate applicability of HLA-targeted FACS sorting after pregnancy by separating chimeric maternal cells from child umbilical cord mononuclear cells. Targeting allelic differences with anti-HLA mAbs with FACS sorting allows maximal enrichment of viable microchimeric cells from a background cell population. The current methodology enables reliable microchimeric cell detection and separation in clinical specimens.

Diverse patterns of T-cell response against multiple newly identified human Y chromosome-encoded minor histocompatibility epitopes.

PURPOSE: Donor T cells respond to minor histocompatibility antigens (mHA), resulting in both graft-versus-host disease and graft versus leukemia after allogeneic hematopoietic stem cell transplantation. Because relatively few mHAs are known, we developed a new approach to predict and subsequently validate candidate mHA. EXPERIMENTAL DESIGN: We developed an algorithm based on genetic disparities between Y chromosome-encoded and X chromosome-encoded proteins and known requirements for binding to HLA class I molecules to predict Y chromosome-derived, HLA A*0201-restricted peptides (HY) and ranked peptides based on potential immunogenicity. We evaluated T-cell responses to 41 candidate peptides in 28 male recipients with female donors (FM), 22 male recipients with male donors (MM), and 26 normal individuals. All patients and donors were HLA A*0201 positive. RESULTS: Thirteen peptides derived from five proteins elicited significantly greater T-cell responses in FM patients compared with MM patients and in normal females compared with normal males. Six peptides were more immunogenic than the only previously known HLA A*0201-restricted Y-encoded mHA. Twenty-seven of 28 FM patients responded to at least one HY peptide, but despite a common Y chromosome mismatch and expression of HLA A*0201, each patient responded to a unique set of peptides. CONCLUSIONS: Novel HLA A*0201-restricted HY epitopes can be predicted and validated in patients after allogeneic hematopoietic stem cell transplantation. Highly diverse patterns of T-cell response against these epitopes have been identified. Prospective monitoring of responses to large panels of immunogenic peptides can facilitate the identification of clinically relevant targets of graft-versus-host disease and graft versus leukemia.

The X in sex: how autoimmune diseases revolve around sex chromosomes.

Recent estimates suggest that autoimmune diseases cumulatively affect 5-10% of the general population worldwide. Although the etiology and pathogenesis remain poorly understood in most cases, similarities between diseases outnumber differences in the initiation and perpetuation of the autoimmune injury. One major example is the predominance of affected women, and perhaps its most intriguing putative mechanism is related to sex chromosomes, based on the recent observation that women with autoimmune diseases manifest a higher rate of circulating leukocytes with a single X chromosome. In a complementary fashion, there have been several reports on a role of X chromosome gene dosage through inactivation or duplication in autoimmunity. It is important not to overlook men with autoimmune diseases, who might manifest a more frequent loss of the Y chromosome in circulating leukocytes. Taken together, sex chromosome changes might constitute the common trait of autoimmunity.

Identification of HLA class II-restricted H-Y-specific T-helper epitope evoking CD4+ T-helper cells in H-Y-mismatched transplantation.

BACKGROUND: Stem-cell grafts between HLA-identical siblings are less likely to succeed when there is a sex mismatch. This lack of success can be interpreted as enhanced activity directed against minor histocompatibility antigens encoded by the Y chromosome (H-Y). So far, in man, only cytotoxic T lymphocytes (CTLs) specific for several minor histocompatibility antigens have been reported. We aimed to identify and clarify the role of MHC class II-restricted H-Y-specific T-helper cells in these transplant settings. METHODS: H-Y-specific MHC class II-restricted CD4+ T cells were isolated from blood of a female patient who rejected an HLA-identical male stem-cell transplant. By molecular cloning of H-Y genes and functional T-helper experiments, we elucidated antigen specificity and the functional properties of these H-Y-specific T-helper cells. FINDINGS: CD4+ T-helper cells recognise the Y gene-encoded peptide VIKVNDTVQI presented by HLA-DRbeta3*0301. These T-helper cells mature dendritic cells and enhance expansion of minor histocompatibility antigen-specific MHC class I-restricted CD8+ CTLs. INTERPRETATION: Characterisation of an MHC class II-restricted H-Y epitope that evoked CD4+ T-helper responses adds a novel cellular component to the alloimmune response against Y chromosome-encoded minor histocompatibility antigens. This component completes the H-Y-directed alloimmune response and aids understanding of the poorer outcome of sex-mismatched transplants.