Fetal gut laser microdissection in combination with RNA preamplification enables epithelial-specific transcriptional profiling.

Laser microdissection (LMD) technology enables highly specific gene expression analyses of biologically relevant questions at cell- or tissue-specific resolution. Nevertheless, specific cell types are often limited in quantity (i.e. fetal tissue), making high quality RNA extraction and subsequent gene expression approaches via common reverse transcriptase-quantitative PCR (RT-q-PCR) challenging. In the case of fetal gut epithelia representing immune modulatory interphases gene expression analysis with common RT-q-PCR is limited to a few genes (<10). To circumvent these limitations we provide a workflow using laser microdissection of 1.5Mioµm(2) dissected area of murine fetal intestinal epithelial cells (IEC) from fetal ileum and colon with subsequent RNA isolation, whole transcriptome preamplification (WTA) and gene expression analysis by microarray and quantitative PCR (qPCR). This workflow allows simultaneous analyses of global (microarrays) and targeted gene expression (qPCR) and consequently increases the number of measurable genes up to 25-fold by qPCR. It is suitable for cryosections from many tissues and species in order to evaluate in utero biological effects on specific effector sites.

Donor CD4 T cells convert mixed to full donor T-cell chimerism and replenish the CD52-positive T-cell pool after alemtuzumab-based T-cell-depleted allo-transplantation.

Donor lymphocyte infusions (DLI) are used to resolve mixed T-cell chimerism (TCC) after allo-SCT despite a substantial risk of GVHD. We analyzed the impact of prophylactic CD8-depleted (CD8(depl)) DLI in 20 recipients of anti-CD52 alemtuzumab in vivo T-cell-depleted allografts with declining donor TCC after day +60. A total of 13 patients received CD8(depl) DLI and 7 patients did not. All but one of the DLI patients converted to complete donor T-cell chimeras, whereas only one non-DLI patient converted spontaneously. DLI induced transient acute GVHD in five and extensive chronic GVHD in two patients. These data suggest the use of CD8(depl) DLI as an effective treatment for mixed TCC, particularly in patients at high risk for GVHD. We also observed that the majority of reconstituting donor-derived T cells after alemtuzumab conditioning were CD52-negative. CD8(depl) DLI significantly increased the proportion of CD52-positive CD4 T cells, whereby their beneficial effect on reconstituting the post-transplant T-cell repertoire was shown.