Immunogenicity of a rheumatoid arthritis protective sequence when acquired through microchimerism

HLA class II genes provide the strongest genetic contribution to rheumatoid arthritis (RA). HLA-DRB1 alleles encoding the sequence DERAA are RA-protective. Paradoxically, RA risk is increased in women with DERAA+ children born prior to onset. We developed a sensitive qPCR assay specific for DERAA, and found 53% of DERAA−/− women with RA had microchimerism (Mc; pregnancy-derived allogeneic cells) carrying DERAA (DERAA-Mc) vs. 6% of healthy women. DERAA-Mc quantities correlated with an RA-risk genetic background including DERAA-binding HLA-DQ alleles, early RA onset, and aspects of RA severity. CD4+ T cells showed stronger response against DERAA+ vs. DERAA− allogeneic cell lines in vitro, in line with an immunogenic role of allogeneic DERAA. Results indicate a model where DERAA-Mc activates DERAA-directed T cells that are naturally present in DERAA−/− individuals and can have cross-reactivity against joint antigens. Moreover, we provide an explanation for the enigmatic observation that the same HLA sequence differentially affects RA risk through Mendelian inheritance vs. microchimeric cell acquisition.

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.