The Ly-6 locus comprises a family of Ly-6 proteins in the mouse, including Ly-6C and Ly-6G. Ly-6C is a 131-amino acid phosphatidylinositol-linked murine cell surface protein that belongs to the Ly-6 gene family (1). Previous studies have shown that Ly-6C is expressed on subsets of T lymphocytes, thymocytes, activated B lymphocytes, and late myeloid/granulocytic precursors (2–6). The two alleles of Ly-6C, Ly-6C.1 and Ly-6C.2, result in differential expression of this protein on subpopulations of CD4+ T cells (4).
Ly-6G, another member of the Ly-6 family, is expressed on granulocytes, a subset of eosinophils, and transiently during developmental stages of monocytes (7–11). Its expression has been studied primarily using the monoclonal antibody (mAb) RB6-8C5 (anti Gr-1), but other monoclonal antibodies (mAbs) have also been developed against this antigen (Ag), including NIMPR-14 (9). Expression of Ly-6G has also recently been described on plasmacytoid dendritic cells (12, 13).
The RB6-8C5 mAb has previously been suggested to cross-react weakly with Ly-6Chi cells in murine bone marrow (BM) and with Ly-6C-transfected EL4J cells (8). This potential cross-reactivity of an anti-Ly-6G mAb with the Ly-6C Ag has been generalized to all hematopoietic cell types and now appears in the recent literature using the terminology Ly-6G/C (12, 14, 15).
Previous unpublished observations in our laboratory have failed to show evidence of anti-Ly-6G staining on Ly-6C+ T cells. To systematically examine the presence or absence of cross-reactivity of anti-Ly-6G mAbs with Ly-6C Ags, two-, three- and four-color flow cytometric analyses of Ly-6G and Ly-6C expression were performed on murine splenocytes, thymocytes, and BM cells using a variety of anti-Ly-6G and anti-Ly-6C mAbs.
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Ly-6 molecules comprise a family of low-molecular-weight phosphatidylinositol anchored cell surface glycoproteins (26, 27). Genes for the Ly-6 proteins in mice are found on chromosome 15 (28). Many Ly-6 molecules, including Ly-6C and Ly-6G, possess conserved domains containing cysteine residues. However, heterogeneity between Ly-6C and Ly-6G is observed in the cysteine spacing within these domains, imparting structural differences between these molecules (26). Amino acid sequencing shows Ly-6G and Ly-6C differ by at least 27 amino acids (29). The extent of homology between Ly-6 molecules has been quantitated using optical alignment techniques. Ly-6G and Ly-6C amino acid sequences have optical alignment standard deviations of 27.1, much greater than the three standard deviations necessary to regard a statistical difference in structure (26). Therefore, it is likely that Ly-6G and Ly-6C exhibit enough structural differences to possess unique epitopes.
A previous study suggested that the mAb RB6-8C5, specific for the Ly-6G Ag, weakly stains Ly-6Chi cells in BM (8). This has been interpreted as cross-reactivity of the RB6-8C5 mAb with the Ly-6C Ag. Perhaps because of this report, the molecule to which RB6-8C5 binds has been called Ly-6G/C, and this terminology has been perpetuated in the recent literature (12, 14, 15).
The two-, three-, and four-color flow cytometric experiments reported herein indicate that mAbs specific for Ly-6G, including RB6-8C5, do not show any binding to many cell populations that express moderate to high levels of Ly-6C. These Ly-6C+ cell populations, which are Ly-6G−, include CD8+ splenocytes, CD8 SP and DN thymocytes, and CpG ODN-activated B cells in both Ly-6.1 and Ly-6.2 strains, as well as CD4+ splenocytes and CD4 SP thymocytes in Ly-6.2 strains. The lack of cross-reactivity was demonstrated using a variety of anti-Ly-6C and anti-Ly-6G mAbs, including an Ly-6C.2 allele-specific mAb (143).
Two BM populations were identified that stain with Ly-6C (Fig. 6). One of these is clearly also positive for Ly-6G. The other population (Ly-6Chi) shows slight positivity with anti-Ly-6G mAbs with certain anti-Ly-6C and anti-Ly-6G fluorochrome combinations. This population is comprised of macrophage and dendritic cell precursors (unpublished observations), and cells of the monocytic lineage have previously been reported to express low levels of Ly-6G (10, 11). Thus, this slight shift could represent detection of true, low-level Ly-6G expression on this population that is apparent with certain fluorochrome-mAb conjugates. It is also formally possible that the observed shift could be the result of anti-Ly-6G cross-reactivity with Ly-6C epitopes expressed on this cell population; however, this explanation is unlikely based on the complete lack of anti-Ly-6G/C cross-reactivity observed in other Ly-6C+ hematopoietic cell types. Alternatively, it is possible that an isoform of Ly-6C is expressed on these cells that is not found on T or B cells, and this particular isoform allows cross-reactivity with the anti-Ly-6G mAbs. To date, there is no published information indicating the presence of more than one Ly-6C gene, or evidence for multiple pathways of post-transcriptional or post-translational modification of the Ly-6C gene product. Therefore, in light of the lack of staining of many other Ly-6C+ populations with anti-Ly-6G, it is likely that the BM cells which stain with anti-Ly-6G mAbs actually express Ly-6G and Ly-6C as independent molecules.
An additional cell population has recently been identified that stains with both Ly-6C and Ly-6G mAbs (12, 13). These cells, termed plasmacytoid dendritic cells, comprise a rare population found in many hematopoietic tissues, including spleen and lymph node. The experiments in this study do not address Ly-6C and Ly-6G expression on this population; however, the same arguments hold for these dual staining populations as for the BM populations that stain with both anti-Ly-6C and anti-Ly-6G mAbs.
In conclusion, no evidence for cross-reactivity between Ly-6C and Ly-6G is identified on B or T cells from Ly-6C.1 and Ly-6C.2 strains of mice. Previously published molecular data also indicate that two separate genes with quite different amino acid sequences exist for these molecules, supporting the existence of unique epitopes on the two molecules. Taken together, these results question the use of the Ly-6G/C nomenclature, and suggest that epitopes recognized by anti-Ly-6G Abs should simply be designated as Ly-6G.