Endogenous expansion of regulatory T cells leads to long-term islet graft survival in diabetic NOD mice.

Donor pancreatic lymph node cells (PLNC) protect islet transplants in Non-obese diabetic (NOD) mice. We hypothesized that induced FoxP3(+) regulatory T cells (Tregs) were required for long-term islet engraftment. NOD or NOD.NON mice were treated with ALS (antilymphocyte serum) and transplanted with NOR islets +/-PLNC (5 × 10(7) ). In vivo proliferation and expansion of FoxP3(+) Tregs was monitored in spleen and PLN from ALS- and ALS/PLNC-treated recipient mice. Anti-CD25 depletion was used to determine the necessity of Tregs for tolerance. FoxP3(+) numbers significantly increased in ALS/PLNC-treated recipients compared to ALS-treated mice. In ALS/PLNC-treated mice, recipient-derived Tregs localized to the transplanted islets, and this was associated with intact, insulin-producing ß cells. Proliferation and expansion of FoxP3(+) Tregs was markedly increased in PLNC-treated mice with accepted islet grafts, but not in diabetic mice not receiving PLNC. Deletion of Tregs with anti-CD25 antibodies prevented islet graft tolerance and resulted in rejection. Adoptive transfer of Tregs to secondary NOD.scid recipients inhibited autoimmunity by cotransferred NOD effector T cells. Treg expansion induced by ALS/PLNC-treatment promoted long term islet graft survival. Strategies leading to Treg proliferation and localization to the transplant site represent a therapeutic approach to controlling recurrent autoimmunity.

Novel signaling interactions between proteinase-activated receptor 2 and Toll-like receptors in vitro and in vivo.

Toll-like receptors (TLRs) and proteinase-activated receptors (PARs) function as innate immune biosensors in mucosal epithelial cells (ECs). We previously reported the functional and physical interactions between TLR4 and PAR(2). We have extended these findings herein by showing the cooperation between PAR(2) and TLR2, TLR3, or TLR4 for activation of nuclear factor-kappaB-dependent signaling in mucosal EC lines. In contrast, activation of PAR(2) negatively regulated TLR3-dependent antiviral pathway, blunting the expression of TLR3/interferon regulatory factor-3 (IRF-3)-driven genes, as well as activation of IRF-3 and STAT1. Consistent with these in vitro observations, PAR(2)(-/-) and TLR4(-/-) mice, which were refractory to footpad edema induced by PAR(2) agonist peptide, were protected from mouse-adapted H1N1 influenza A virus-induced lethality when compared to wild-type (WT) mice. These data support and extend our recently described, novel model of PAR(2)-TLR4 "receptor cooperativity" and highlight the complexity of signaling integration between heterologous innate immune biosensors.

Generation and biochemical analysis of human effector CD4 T cells: alterations in tyrosine phosphorylation and loss of CD3zeta expression.

Human effector T cells have been difficult to isolate and characterize due to their phenotypic and functional similarity to the memory subset. In this study, a biochemical approach was used to analyze human effector CD4 T cells generated in vitro by activation with anti-CD3 and autologous monocytes for 3 to 5 days. The resultant effector cells expressed the appropriate activation/differentiation markers and secreted high levels of interferon gamma (IFN-gamma) when restimulated. Biochemically, effector CD4 T cells exhibited increases in total intracellular tyrosine phosphorylation and effector-associated phosphorylated species. Paradoxically, these alterations in tyrosine phosphorylation were concomitant with greatly reduced expression of CD3zeta and CD3epsilon signaling subunits coincident with a reduction in surface T-cell receptor (TCR) expression. Because loss of CD3zeta has also been detected in T cells isolated ex vivo from individuals with cancer, chronic viral infection, and autoimmune diseases, the requirements and kinetics of CD3zeta down-regulation were examined. The loss of CD3zeta expression persisted throughout the course of effector T-cell differentiation, was reversible on removal from the activating stimulus, and was modulated by activation conditions. These biochemical changes occurred in effector T cells generated from naive or memory CD4 T-cell precursors and distinguished effector from memory T cells. The results suggest that human effector T-cell differentiation is accompanied by alterations in the TCR signal transduction and that loss of CD3zeta expression may be a feature of chronic T-cell activation and effector generation in vivo. (Blood. 2001;97:3851-3859)

Differential role of CTLA-4 in regulation of resting memory versus naive CD4 T cell activation.

Regulation of peripheral T cell responses is critical for preserving self tolerance. Memory T cells have a lower threshold for activation through the TCR and are thought to be less dependent on costimulation than naive T cells, suggesting a requirement for more stringent regulation of memory T cells. We have recently shown that CD4 engagement apart from the TCR results in the inactivation of memory, but not naive, CD4 T cells. We show here that this inhibition requires ligation of CTLA-4, in that blocking CTLA-4-B7 interactions restores memory CD4 T cell responsiveness. Early signaling through CTLA-4 is possible because resting memory, but not naive, CD4 T cells contain intracellular stores of CTLA-4 that are continuously recycled between the cytoplasm and the cell surface. This mechanism ensures that low intensity TCR engagements, which are thought to be important for peripheral T cell longevity, do not cause memory T cell activation but instead raise their threshold for costimulatory signals. This may give memory T cells an extended lifespan with a reduced risk of inappropriate activation.

Rat class III Fc gamma receptor isoforms differ in IgG subclass-binding specificity and fail to associate productively with rat CD3 zeta.

Several new rat class III Fc gamma R isoforms are described here, extending the genetic complexity of this receptor family and further distinguishing rat CD16 from mouse CD16, represented by only one receptor isoform, and human CD16, represented by only two isoforms. RNase protection assays reveal that three rat tumor cell lines--RBL-1 basophilic leukemia cells, RM-SV1 macrophages, and CRNK-16 NK cells--all coordinately express multiple and probably identical rtFc gamma RIII-related transcripts in similar relative proportions but at significantly different levels. These results indicate that no single isoform predominates in these cell types but that the overall level of rtFc gamma RIII-related transcripts is differentially regulated. Two of the rtFc gamma RIII isoforms found to have extensive amino acid sequence differences in their second extracellular (EC2) domains are shown to bind rat and mouse IgG subclasses differently. This result suggests that the receptor isoform diversity in this species may function as a mechanism for extending the IgG-binding capacity of rat leukocytes. Cloned cDNA for the rat CD3 zeta protein was also isolated in this study and its ability to augment surface expression of class III Fc gamma R was tested by rosetting of cDNA-transfected COS cells. Like the structurally homologous mouse CD3 zeta, rat CD3 zeta fails to promote surface expression of Fc gamma RIII, sharply contrasting the efficient receptor expression produced by human CD3 zeta. Variations in the transmembrane amino acid sequences correlate with the divergent capacities of these CD3 zeta molecules to augment receptor expression. The high levels of CD3 zeta message expressed in rat NK cells may indicate that other unidentified hetero-subunits are required for assembly of rat CD3 zeta into functional CD16 receptors.

Rat CD16 is defined by a family of class III Fc gamma receptors requiring co-expression of heteroprotein subunits.

Rat CD16 is defined here by a family of highly homologous class III Fc gamma receptor isoforms. Northern blot and polymerase chain reaction analysis indicate that multiple rtFc gamma RIII alpha isoforms are expressed by rat NK and macrophages contrasting the expression of a single class III receptor isoform by human and mouse NK and macrophages. Analysis of genomic Southern blots and splice variants identified by polymerase chain reaction suggests the existence of several homologous rat Fc gamma RIII alpha genes organized similar to human and mouse class III receptor genes. All rat Fc gamma RIII alpha isoform protein sequences have conventional transmembrane insertion sequences containing the unique LFAVDTGL motif conserved in all other class III Fc gamma and Fc epsilon RI alpha receptor sequences. A model is proposed for the protein-protein interactions between this sequence and the transmembrane sequences of two heteroprotein subunits, Fc epsilon RI gamma and CD3 zeta, known to interact with human and mouse class III receptors. Rat NK, monocytes, and neutrophils were all found to express transcripts for both of these subunits, whereas rat macrophages express only Fc epsilon RI gamma subunit transcripts. Furthermore, the Fc epsilon RI gamma subunit was found to promote the cell surface expression of rtFc gamma RIII alpha isoforms in transfected COS cells.