+ T cells distinguishes between Treg and pathogenic cellular populations that secrete proinflammatory cytokines such as IFNc and IL-17. These latter cell populations are increased, with a concomitant decrease in the CD4 + CD25 + CD39 + Tregs, in the peripheral blood of patients with renal allograft rejection. We conclude that the ectonucleotidase CD39 is a useful and dynamic lymphocytes surface marker that can be used to identify different peripheral blood T cell-populations to allow tracking of these in health and disease, as in renal allograft rejection.

The production of synthetic MHC-peptide tetramers has revolutionized cellular immunology by revealing enormous CD8+ T cell expansions specific for peptides from various pathogens. A feature of these reagents, essential for their staining function, is that they bind T cells with relatively high avidity. This could, theoretically, promote cross-reactivity with irrelevant T cells leading to overestimates of epitope-specific T cell numbers. Therefore, we have investigated the fine specificity of CTL staining with these reagents for comparison with functional data. Using a panel of CTL clones with distinct fine specificity patterns for analogs of an HLA-B8-binding EBV epitope, together with B8 tetramers incorporating these peptides, we show a very good correlation between tetramer staining and peptide activity in cytotoxicity assays. Significant staining only occurred with tetramers that incorporate strong stimulatory agonist peptides and not weak agonists that are unlikely to induce full T cell activation at physiological levels of presentation. In almost every case where a peptide analog had >10-fold less activity than the optimal EBV peptide in cytotoxicity assays, the corresponding tetramer stained with >10-fold less intensity than the EBV epitope tetramer. Furthermore, by examining an EBV-specific clonotypic T cell expansion in EBV-exposed individuals, we show similar fine specificity in tetramer staining of fresh peripheral T cells. Collectively, our data demonstrate the exquisite specificity of class I MHC-peptide tetramers, underlining their accuracy in quantifying only those T cells capable of recognizing the low levels of cell surface peptide presented after endogenous Ag processing.

Various studies have shown that major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTL) can be isolated from lymph nodes draining sites of cutaneous infection with herpes simplex virus type 1 (HSV-1). Invariably, detection of this cytolytic activity appeared to require some level of in vitro culture of the isolated lymph node cells, usually for 3 days, in the absence of exogenous viral antigen. This in vitro "resting" period was thought to represent the phase during which committed CD8 ؉ T cells become "armed" killers after leaving the lymph nodes and prior to their entry into infected tissue as effector CTL. In this study we reexamined the issue of CTL appearance in the HSV-1 immune response and found that cytolytic activity can be isolated directly from draining lymph nodes, although at levels considerably below those found after in vitro culture. By using T-cell receptor elements that represent effective markers for class I-restricted T cells specific for an immunodominant glycoprotein B (gB) determinant from HSV-1, we show that the increase in cytotoxicity apparent after in vitro culture closely mirrors the expansion of gB-specific CTL during the same period. Taken together, our results suggest that HSV-1-specific CTL priming does not appear to require any level of cytolytic machinery arming outside the lymph node compartment despite the absence of any detectable infection within that site.Major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL) can play a critical role in antiviral immune responses (40). These T cells facilitate viral clearance by directly lysing target cells harboring productive virus infection. In the case of the human immune response to herpes simplex virus type 1 (HSV-1), CD8 ϩ T cells can be isolated from infected individuals (32). In addition, the recent identification of the HSV-encoded ICP47 protein, which blocks class I-restricted peptide presentation, suggests that CTL lysis plays an important role during the antiviral response (11,15,39). In mouse model systems, CD8 ϩ T cells can be isolated from lymph nodes draining the site of cutaneous primary infection, and these T cells effectively protect animals against subsequent infection (2, 3, 13, 22, 28). However, unlike many primary antiviral responses, T cells isolated from lymph nodes draining sites of cutaneous HSV-1 infection did not appear to kill infected target cells without being subjected to a period of in vitro culture, usually in the absence of exogenous antigen (6,27,30). This culture period was thought to reflect a need for extralymphoid differentiation into armed cytotoxic effectors capable of dealing with the peripheral infection. Such a proposal is consistent with the notion that while CTL precursors are activated within the draining lymph nodes, the cytotoxic effectors are required only within the actual sites harboring infective virus, in this case the infected footpads.We have examined the CD8 ϩ CTL response to footpad infection with HSV-1. This response is d...

In this study, we demonstrate the expression of ligands for human NKG2D on porcine cell lines of endothelial and epithelial origin, islet cell clusters and rejecting kidney. HuNK cells were activated to kill pig cells expressing NKG2D ligands, and cytotoxicity was inhibited by antibody blockade of NKG2D. A previous study identified pULBP1 as the principal ligand for human NKG2D on pig aortic endothelial cells. In the current study, renal epithelial and intestinal endothelial cells each expressed high surface levels of pULBP1, but binding of soluble recombinant NKG2D and NKG2D-dependent cytotoxicity against these cells persisted after the enzymatic removal of pULBP1, strongly suggesting the presence of at least one additional functional ligand for human NKG2D in these cell types.

SummaryThe human cytomegalovirus (HCMV) UL16 gene encodes a glycoprotein that interferes with the immune response to the virus-infected cell. In vitro , UL16 interacts with MICB and ULBPs that are ligands for the stimulatory receptor NKG2D, expressed on NK cells and CD8 + + + + T cells. UL16 expression has been shown to promote intracellular accumulation of MICB, ULBP1 and 2 and thus, interfere with the immune response to HCMV-infected cells. The mechanism that has been suggested for UL16-mediated MICB downmodulation is retention in the ER. Here, we studied the intracellular localization and maturation of UL16 and MICB in HCMV-infected cells and transfectant systems. UL16 trafficked through the ER, TGN and progressed to the plasma membrane, after which the protein was internalized. Strikingly, UL16 was also observed in the inner nuclear membrane. MICB was also localized in the TGN in HCMV-infected cells. These data suggest that MICB trafficking might be affected after its transit through the ER.

No abstract

Subsets of CD8 T cells express receptors that are critical in regulating the activity of NK cells. To characterize the expression of these receptors on CD8 T cells we made use of transgenic mice that express a H-2Kb restricted TCR specific for the immunodominant epitope located within the HSV-1 glycoprotein B (gB). Few naive gB-specific T cells express Ly49 or CD94/NKG2 receptors. Following acute infection of C57BL/6 mice with either HSV-1 or a recombinant influenza virus that encodes the gB determinant, gB-specific T cells showed a dramatic upregulation of CD94/NKG2 receptors. Moreover, gB-specific CD8 T cells that expressed CD94/NKG2 receptors were also found to express another NK receptor, KLRG1. We established that while Ag-stimulated gB-specific CD8 T cells primarily express inhibitory isoforms of CD94/NKG2 receptors, these cells remain capable of producing gammaIFN upon peptide stimulation. While peak CD94/NKG2 expression on gB-specific cells was reached 2-3 days following infection, it remained elevated beyond 60 days post-infection with either HSV-1 or a gB-expressing recombinant influenza virus. The data imply that the prolonged expression was not due to persistence of replicating virus and suggest that while recognition of the cognate Ag is necessary to trigger expression of CD94/NKG2 receptors, it is not required for their continued expression on memory T cells.

Differential protein glycosylation in the donor and recipient can have profound consequences for transplanted organs, as evident in ABO‐incompatible transplantation and xenotransplantation. In this study, we investigated the impact of altered fucosylation on graft acceptance by using donor mice overexpressing human α1,2‐fucosyltransferase (HTF). Skin and heart grafts from HTF transgenic mice were rapidly rejected by otherwise completely matched recipients (median survival times 16 and 14 days, respectively). HTF skin transplanted onto mice lacking T and B cells induced an natural killer cell–mediated innate rejection crisis that affected 50–95% of the graft at 10–20 days. However, in the absence of adaptive immunity, the residual graft recovered and survived long‐term (>100 days). Experiments using “parked” grafts or MHC class II‐deficient recipients suggested that indirect rather than direct antigen presentation plays a role in HTF skin graft rejection, although the putative antigen(s) was not identified. We conclude that altered glycosylation patterns on donor tissue can trigger a powerful rejection response comprising both innate and adaptive components. This has potential implications for allotransplantation, in light of increasing recognition of the variability of the human glycome, and for xenotransplantation, where carbohydrate remodeling has been a lynchpin of donor genetic modification.