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Dendritic cells (DC) represent key regulators of the immune system, yet their development from hemopoietic precursors is poorly defined. In this study, we describe an in vitro system for amplification of a Flt3(+)CD11b(+) progenitor from mouse bone marrow with specific cytokines. Such progenitor cells develop into both CD11b(+) and CD11b(-) DC, and CD8alpha(+) and CD8alpha(-) DC in vivo. Furthermore, with GM-CSF, these progenitors synchronously differentiated into fully functional DC in vitro. This two-step culture system yields homogeneous populations of Flt3(+)CD11b(+) progenitor cells in high numbers and allows monitoring the consecutive steps of DC development in vitro under well-defined conditions. We used phenotypic and functional markers and transcriptional profiling by DNA microarrays to study the Flt3(+)CD11b(+) progenitor and differentiated DC. We report here on an extensive analysis of the surface Ag expression of Flt3(+)CD11b(+) progenitor cells and relate that to surface Ag expression of hemopoietic stem cells. Flt3(+)CD11b(+) progenitors studied exhibit a broad overlap of surface Ags with stem cells and express several stem cell Ags such as Flt3, IL-6R, c-kit/SCF receptor, and CD93/AA4.1, CD133/AC133, and CD49f/integrin alpha(6). Thus, Flt3(+)CD11b(+) progenitors express several stem cell surface Ags and develop into both CD11b(+) and CD11b(-) DC, and CD8alpha(+) and CD8alpha(-) DC in vivo, and thus into both of the main conventional DC subtypes.

Dendritic cells (DCs) are potent antigen-presenting cells with a pivotal role in antigen-specific immune responses. Here, we found that the helix-loop-helix transcription factor Id2 is up-regulated during DC development in vitro and crucial for the development of distinct DC subsets in vivo. Id2-/- mice lack Langerhans cells (LCs), the cutaneous contingent of DCs, and the splenic CD8alpha+ DC subset is markedly reduced. Mice deficient for transforming growth factor (TGF)-beta also lack LCs, and we demonstrate here that, in DCs, TGF-beta induces Id2 expression. We also show that Id2 represses B cell genes in DCs. These findings reveal a TGF-beta-Id2 signaling pathway in DCs and suggest a mechanism by which Id2 affects the lineage choice of B cell and DC progenitors.

Rat natural killer (NK) cells recognize MHC-I molecules encoded by both the classical (RT1-A) and non-classical (RT1-C/E/M) MHC class I (MHC-I) regions. We have identified a receptor, the STOK2 antigen, which belongs to the Ly-49 family of killer cell lectin-like receptors, and we have localized the gene encoding it to the rat natural killer cell gene complex. We have also shown that it inhibits NK cytotoxicity when recognizing its cognate MHC-I ligand RT1-A1c on a target cell. This is the first inhibitory Ly-49-MHC-I interaction identified in the rat and highlights the great similarity between rat and mouse Ly-49 receptors and their MHC ligands. However, the mode of rat NK-cell recognition of target cells indicates that positive recognition of allo-MHC determinants, especially those encoded by the RT1-C/E/M region, is a prevalent feature. NK cells recruited to the peritoneum as a consequence of alloimmunization display positive recognition of allodeterminants. In one case, NK cells activated in this way have been shown to be specific for the immunizing, non-classical class I molecule RT1-Eu. These findings show that allospecific NK cells sometimes show features reminiscent of the adaptive immune response.

Polyclonal rabbit anti-idiotypic (Ab2) antibodies raised against the antiprogesterone mAb DB3 (Ab1) were used to induce an Ab3 antiprogesterone response in BALB/c mice. While the affinity of Ab3 sera for progesterone was 10-50-times lower than that of DB3, their steroid-binding specificity showed considerable similarity to DB3. Two immunoglobulin M (IgM) Ab3 monoclonal antibodies (mAbs), 1A4 and 3B11, were obtained, both of which bound progesterone conjugated to bovine serum albumin (progesterone-BSA). 1A4 also bound free progesterone, although with low affinity and very broad cross-reactivity. Like DB3, 1A4 is encoded by a heavy-chain variable region (VH) gene segment from the small VGAM3.8 family, a restriction that is characteristic of antibodies raised against progesterone-11alpha-BSA. In contrast, 3B11 binds progesterone-11alpha-BSA but not free progesterone and is encoded by an unrelated VH gene from the J558 family. The light chain variable region (VL) of 1A4 lacks the intradomain disulphide bridge owing to replacement of CysL23 by Tyr. Both the 1A4 and 3B11 heavy chains have extremely short complementarity determining region (CDR) H3 loops, comprising three and four amino acids, respectively. Modelling of the combining site of 1A4 from the X-ray crystallographic structure of DB3 indicates that the short H3 loop is a major factor in the loss of affinity and specificity for steroid.

The QuikChangeTM protocol is one of the simplest and fastest methods for site-directed mutagenesis, but introduces mutations at only one site at a time, and requires two HPLC-purified complementary oligonucleotides. Here, we describe that this method can be used with non-overlapping oligonucleotides. By doing this, two separate sites can be mutagenised simultaneously, or money can be saved by using a second 'standard' oligonucleotide. By a further modification, we have also used the QuikChangeTM approach to exchange DNA sequences between closely related genes.