Reagents
Immunochemistry microscopy on frozen spleen section
of at low magnification from a Xenopus juvenile double
stained for IgM+ (blue) and proliferating BrdU+ (brown) cells.
(From Du Pasquier et al., 2000)
mAbs allowing the phenotypic characterization of various leukocyte populations include: markers of thymocytes and peripheral T-cell subpopulations such as the pan T cell XT-1 mAb (Nagata, 1985); two mAbs against a CD8-equivalent T cell co-receptor (Flajnik et al., 1991; Ibrahim et al, 1991); a mAb that recognizes a CD5 epitope (Jürgens et al., 1995), and another that detects a CD45-like molecule (Barritt & Turpen, 1995). All these mAb reagents are available in our laboratory, as are mAbs that detect MHC class I and class II molecules (Flajnik et al., 1990; 1991).
The mAb 1F8 identifies a surface protein specifically expressed by a NK-like lymphoid population (Horton et al., 2000) that remains present in T-cell deficient frogs (thymectomized) and that kill MHC class I-negative tumor targets. NK cells emerge as a small population in the spleen of metamorphosing larvae one week after the first appearance of surface MHC class Ia (Horton et al., 2003).
Three immunoglobulin isotypes have been described in X. laevis (Hsu, 1998; Du Pasquier et al., 2000): IgM, IgY and IgX. X. laevis IgM is clearly homologous to mammalian IgM. IgY is believed to be a modern equivalent of an ancient protein that gave rise to mammalian IgE and IgG. IgX is functionally analogous to mammalian IgA but structurally is more similar to X. laevis IgM (Mussmann et al., 1996). IgM and IgX appear to be thymus independent isotypes, whereas IgY is produced during T-dependent immune responses. Mabs specific for the three different heavy chains as well as for Light chain isotypes have been characterized (Table 1, Bleicher & Cohen, 1981; Hsu et al., 1984). The various X. laevis cDNA libraries available include: those from adult, metamorphic, and larval total splenocytes (from naïve and immunized animals), those form purified adult NK and CD8 T cell population, and those from the 15/0 and ff-2 lymphoid tumors. Finally, there is available a whole variety of cDNA clones of immunologically relevant genes useful to generate molecular probes. EST and BAC libraries are also available. (ref)
Expression Pattern of X. laevis Surface Markers Detectable With Currently Available mAbs
| Markers (mAbs) | Expression Pattern |
|---|---|
| CD8 (AM22, F17) | Larval and adult thymocytes (70-80%) and T-cells (about 20% of splenocytes). All lymphoid tumor lines. |
| CTX (X71, 1S9.2) | Larval and adult thymocytes (60-70%); no consistent expression in peripheral lymphocytes. All lymphoid tumor lines, gut epithelial tissue. |
| XT1 (XT1) | Most, but not all, larval and adult T-cells; earliest marker of thymocytes. All lymphoid tumor lines |
| MHC class I (TB17) | Ubiquitous in adult. Not consistent expression until metamorphosis. |
| MHC class II (AM20, 14A2) | Thymocytes, B and T-cells (99% of spleen lymphocytes), only B-cells in larvae. |
| CD5 (2B1) | Thymocytes (>95%), T-cells and some activated IgM+ B cells. All lymphoid tumor lines. |
| CD45 (CL21) | T and B cells. All lymphoid tumor lines. |
| NK-like (1F8) | Non-B and non-T, peripheral lymphoid cells. |
| RC47 | Leukocyte lineage from very early stage. Thymic cortex and medulla (>90% of total thymocytes). |
| IgM (10A9, 6.16) | Larval and adult B cells. |
| IgY (11D5) | Some larval and adult B cells. |
| IgX (410D9) |
Some larval and adult B cells, especially in the gut. |
| Light chain (1E9, 13B2) | Larval and adult B cells. |
* No mAbs specific for CD4 or TCR have been described so far.