Our recent studies demonstrated high avidity binding of RTLs to macrophages, dendritic cells and B cells, and such RTL “armed” myeloid cells (but not B cells) could tolerize T cells specific for the RTL-bound peptide 43. The current study clearly demonstrates that two-domain MHC-II complexes embodied by RTLs are distinct from the corresponding four-domain complexes, and these two-domain structures deliver

INCB018424 mw tolerogenic rather than activating signals through the cognate TCR. We believe that the RTL-armed APCs are tolerogenic through two possible mechanisms: (i) that the RTLs present on the APC surface can still ligate the TCR of cognate T cells suboptimally as partial agonists; and (ii) the RTLs induce inhibitory cell surface co-inhibitory molecules (e.g. PD-1 or PD-L1/2) and/or secreted inhibitory cytokines (e.g. IL-10) www.selleckchem.com/products/abt-199.html that inhibit T-cell activation in concert with RTL ligation of the TCR, with or without prior processing and re-presentation of RTL-derived antigenic peptide and MHC determinants. Our TCRL Fabs

will be used to further elucidate the in vivo therapeutic pathways of RTL1000 in the humanized DR2-Tg EAE model. RTL342m idiotype-specific TCRLs can be used to both inhibit RTL binding to APC and block RTL association with the TCR, as would be predicted for Fab 2E4. A similar approach can shed light on the functionality of the novel native two-domain structures and address whether they constitute Ag-specific tolerogens that resemble RTLs regulatory pathways. By using our conformational sensitive Fabs we will test our hypothesis that natural RTL-like structures are degradation products of soluble four-domain MHC-II molecules that have undergone

partial enzymatic cleavage. In addition, we are in the process of isolating TCRL Fabs specific for the native DR2–MOG-35-55 complex. Such Fabs will enable us to monitor possible processing and re-presentation of RTL peptides by APCs. In recent years, with the advantage of fluorochrome-labeled MHC-II multimers, there is increased knowledge about specific CD4+ T cells in various inflammatory autoimmune conditions 14, 44–47. T1D patients and at-risk subjects were found to have a significantly higher prevalence of GAD-555-567-specific CD4+ T cells than control selleckchem subjects 48. Our novel TCRL to four- versus two-domain MHC-II–peptide complexes have the potential to selectively recognize APCs presenting disease-inducing or regulatory determinants, respectively, to islet cell-responsive CD4+ T cells during T1D. Similarly, Fabs to four- versus two-domain DR2–MOG-35-55 determinants may be invaluable in localizing and quantifying encephalitogenic versus tolerogenic APC in subjects with MS. RTL1000 and RTL340 constructs were modified for a biotinylated version. In these constructs, a Bir-A tag for biotinylation was introduced to the N-terminus using a 20-aa flexible linker.