Simona Porcellini

Constitutive Endocytosis and Degradation of the Pre-T Cell Receptor

The pre-T cell receptor (TCR) signals constitutively in the absence of putative ligands on thymic stroma and signal transduction correlates with translocation of the pre-TCR into glycolipid-enriched microdomains (rafts) in the plasma membrane. Here, we show that the pre-TCR is constitutively routed to lysosomes after reaching the cell surface. The cell-autonomous down-regulation of the pre-TCR requires activation of the src-like kinase p56lck, actin polymerization, and dynamin. Constitutive signaling and degradation represents a feature of the pre-TCR because the γδTCR expressed in the same cell line does not exhibit these features. This is also evident by the observation that the protein adaptor/ubiquitin ligase c-Cbl is phosphorylated and selectively translocated into rafts in pre-TCR– but not γδTCR-expressing cells. A role of c-Cbl–mediated ubiquitination in pre-TCR degradation is supported by the reduction of degradation through pharmacological inhibition of the proteasome and through a dominant-negative c-Cbl ubiquitin ligase as well as by increased pre-TCR surface expression on immature thymocytes in c-Cbl–deficient mice. The pre-TCR internalization contributes significantly to the low surface level of the receptor on developing T cells, and may in fact be a requirement for optimal pre-TCR function.

GKLF IN THYMUS EPITHELIUM AS A DEVELOPMENTALLY REGULATED ELEMENT OF THYMOCYTE-STROMA CROSS-TALK

Gut-enriched Krüppel-like factor (GKLF) is a transcriptional regulator expressed in differentiated epithelia. We identified GKLF transcript as a regulated element in thymic epithelium of recombinase-deficient mice during thymus development induced by anti-CD3 antibody injection. This treatment recapitulates the organogenetic process depending on productive rearrangement of T cell receptor (TCR) beta gene with thymocytes expansion and acquisition of the CD4+8+ double positive phenotype. In wildtype mice, GKLF is expressed very early in embryogenesis and becomes intensely up-regulated in thymus epithelium at day 18 of gestation when TCR beta expressing cells have selectively expanded and express both CD4 and CD8. The results presented here suggest that thymocytes may regulate GKLF transcriptionally in the cortical epithelium at the developmental check-point controlled by TCR beta gene rearrangement. Furthermore, GKLF expression in hematopoietic stroma might suggest the thus far uncharacterised participation of this factor in hematopoiesis.

Molecular and cellular aspects of induced thymus development in recombinase‐deficient mice

Thymus development and microenvironment organization require stage‐ and site‐specific cross‐talk between thymocyte and stroma. In this study we have used recombinase‐activating gene‐deficient (RAG‐2– / –) mice to analyze regulated gene expression both in thymocytes and stromal cells following injection of anti‐CD3 monoclonal antibodies as inducer of thymus development. We show that IFN‐γ, TNF‐α and lymphotactin are transcriptionally regulated in thymocytes, whereas cytoskeletal keratin 14, IL‐1α and TNF‐α are regulated in the stroma, quantitatively reproducing the variations associated with β selection of thymocytes. In addition, RAG‐2– / – thymus development is associated with entry of epithelial cells into the cell cycle. The histochemical evidence that expanded RAG‐2– / – thymus becomes undistinguishable from wild‐type cortex further suggests that cross‐talk phenomena occurring during β selection of thymocyte are reproduced in this system.

Selective Preservation of Bone Marrow Mature Recirculating but Not Marginal Zone B Cells in Murine Models of Chronic Inflammation

Inflammation promotes granulopoiesis over B lymphopoiesis in the bone marrow (BM). We studied B cell homeostasis in two murine models of T cell mediated chronic inflammation, namely calreticulin-deficient fetal liver chimeras (FLC), which develop severe blepharitis and alopecia due to T cell hyper responsiveness, and inflammatory bowel disease (IBD) caused by injection of CD4+ naïve T cells into lymphopenic mice. We show herein that despite the severe depletion of B cell progenitors during chronic, peripheral T cell-mediated inflammation, the population of BM mature recirculating B cells is unaffected. These B cells are poised to differentiate to plasma cells in response to blood borne pathogens, in an analogous fashion to non-recirculating marginal zone (MZ) B cells in the spleen. MZ B cells nevertheless differentiate more efficiently to plasma cells upon polyclonal stimulation by Toll-like receptor (TLR) ligands, and are depleted during chronic T cell mediated inflammation in vivo. The preservation of mature B cells in the BM is associated with increased concentration of macrophage migration inhibitory factor (MIF) in serum and BM plasma. MIF produced by perivascular dendritic cells (DC) in the BM provides a crucial survival signal for recirculating B cells, and mice treated with a MIF inhibitor during inflammation showed significantly reduced mature B cells in the BM. These data indicate that MIF secretion by perivascular DC may promote the survival of the recirculating B cell pool to ensure responsiveness to blood borne microbes despite loss of the MZ B cell pool that accompanies depressed lymphopoiesis during inflammation.

Recombinase-deficient T cell development by selective accumulation of CD3 into lipid rafts

The pre‐T cell receptor (pre‐TCR) promotes the development of thymocytes with productive rearrangement at the TCR β chain locus by signaling in a ligand‐independent fashion. The TCR β chain associates with the invariant pre‐Tα (pTα) chain, which bears specific charged residues in the extracellular portion mediating pre‐TCR self‐oligomerization. In recombinase‐deficient thymocytes, calnexin (CNX) associated with CD3 chains is inefficiently retained in the endoplasmic reticulum (ER) and weakly expressed in the plasma membrane. Deliberate cross‐linking of CNX/CD3 complexes mimics pre‐TCR signaling. Here, we show that, analogously to the pTα chain, surface CNX is palmitoylated and that CD3 prominently accumulated in lipid rafts upon cross‐linking. Mutant CNX isoforms devoid of ER retention determined pre‐TCR‐like signaling and simulated β selection only when stably translocating CD3 to lipid rafts. Inclusion of the palmitoylated cytoplasmic tail from the pTα chain in recombinant CNX strikingly improved the pre‐TCR‐like signaling efficiency of CNX/CD3 in rafts. This study indicates that lipid rafts in the plasma membrane represent proficient microdomains for the initiation of pre‐TCR signaling, and supports the view that β selection by oligomerized pre‐TCR is implemented by the pTα cytoplasmic tail.

Improved thymopoietic potential in aviremic HIV-infected individuals treated with HAART by intermittent IL-2 administration

Objective: In HIV-positive individuals administration of intermittent interleukin (IL)-2 in addition to highly active antiretroviral therapy (HAART) induces expansion of the peripheral T cell pool with dilution of signal joint T cell receptor excision circles (sjTREC) that cannot be used to measure thymic output. We analysed whether in vitro thymopoiesis could be used to predict in vivo thymic output in IL-2 treated subjects. Design and methods: We correlated the relative variation of peripheral CD4 T cells over 12 months in HIV-positive subjects on HAART or HAART + IL-2 with the mean levels of both sjTREC and T cells developed in chimeric murine foetal thymic organ cultures (FTOC) reconstituted with circulating progenitors. Results: In contrast with HAART treated individuals in which these values were directly correlated, in subjects receiving HAART + IL-2 the increase of CD4 T cells in vivo was correlated to neither sjTREC number nor to reconstitution of FTOC, probably reflecting a main effect of IL-2 in the expansion of the peripheral T cell pool. Nevertheless, addition of IL-2 to HAART determined a significant increase of in vitro thymopoietic potential in individuals with undetectable viraemia. Conclusions: The increased T cell development in vitro after addition of IL-2 to HAART suggests that intermittent IL-2 administration may exert a positive influence on lymphopoiesis. In two subjects with positive viraemia treated with IL-2 we observed reduced in vitro development of T cell precursors suggesting that the positive influence of IL-2 on thymopoiesis could be secondary to the control of viral replication by HAART. These observations provide novel evidence in support of the potential beneficial use of IL-2 in HAART treated individuals.

Regulation of peripheral T cell activation by calreticulin

Porcellini et al. 2006. J. Exp. Med. doi:10.1084/jem.20051519 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20051519%26rft_id%253Dinfo%253Apmid%252F16492806%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%