ISG15 is a 15-kDa protein of unique primary amino acid sequence, which is transcriptionally regulated by interferon (IFN) cx and IFN-f3. Because it is synthesized in many cell types and secreted from human monocytes and lymphocytes, we postulated that ISG15 might act to modulate immune cell function. ISG15 stimulated B-depleted lymphocyte proliferation in a dose-dependent manner with significant proliferation induced by amounts of ISG15 as low as 1 ng/ml (58 pM Although molecular mechanisms underlying biological responses to interferons (IFNs) are only partially dissected, it is thought that they are mediated by the regulated synthesis of induced proteins (1, 2). One of these IFN-induced gene products is ISG15 (3,4). ISG15 is a 15-kDa protein that is transcriptionally regulated by IFN-a or B (5,6). ISG15 is synthesized in mammalian cells as a 17-kDa precursor (pre-ISG15) that is processed by a cellular converting enzyme, cleaving the 8 carboxyl-terminal aa to yield the 157-aa mature ISG15 (4, 7), which is secreted from monocytes and lymphocytes (8). Both native and recombinant ISG15 induce the synthesis and secretion of IFN-'y from B-depleted lymphocytes (9).Based on its cytokine-like properties, it was hypothesized that ISG15 modulates immune effector cell activation and function. To evaluate this, effects on peripheral blood lymphocytes were assessed by [3Hlthymidine incorporation, immunophenotyping, and cytolytic assays. We demonstrate that ISG15 induced production of IFN-,y from T cells, augmented the proliferation of natural killer (NK) cells, and inducedThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.non-major histocompatibility complex-restricted cytolysis of tumor cell targets by NK-derived lymphokine-activated killer (LAK) cells in the absence of detectable levels of interleukin (IL) 2 or IL-12.MATERIALS AND METHODS Materials. B-depleted peripheral blood lymphocytes (PBLs) were purified by passage over a nylon wool column (9). By flow cytometry, this population was 80-85% CD3+, 5-8% CD16+, <2% CD14+, and <2% CD19+. Human recombinant IL-2 (2 x 107 units/mg) was from Hoffmann-LaRoche. Human recombinant IFN-y (2 x 107 units/mg) was from Biogen. A polyclonal antiserum to homogenous ISG15, purified from cytoplasms of IFN-,B-treated Daudi cells, was raised in a New Zealand White rabbit (7). Antigenic specificities of monoclonal antibodies used for flow cytometry included CD3 (Leu 4), CD14 (Leu M3), CD16 (Leu lla), CD19 (Leu 12), and CD56 (NKH-1) (Becton Dickinson).Expression and Purification of ISG15 and pre-ISG15. Human 157-aa ISG15 and the 165-aa pre-ISG15 were expressed and purified from Escherichia coli BL21(DE3) (10, 11). Endotoxin levels were <0.03 endotoxin units (EU)/,tg as measured by limulus amoebocyte lysate assay (sensitivity, 0.01 EU/ml). Heat-denatured ISG15 was ISG15 boiled for 20 min.Functional Assays. Proliferation ([3H]thy...

CD200 (OX-2) is a transmembrane glycoprotein that transmits an immunoregulatory signal through the CD200 receptor (CD200R) to attenuate inflammatory reactions and promote immune tolerance. CD200 expression in the skin has not been described previously. We now report that freshly isolated cells of the murine epidermis contain a subpopulation of major histocompatibility complex (MHC) class II-negative, CD3-negative keratinocytes that are CD200-positive. CD200 expression was accentuated in keratinocytes comprising the outer root sheath of the murine hair follicle (HF). When syngeneic skin grafts were exchanged between gender-matched wild-type (WT) and CD200-deficient C57BL/6 mice, significant perifollicular and intrafollicular inflammation was observed, eventually leading to the destruction of virtually all HF (alopecia) without significant loss of the CD200-negative grafts. Minimal and transient inflammation was observed in WT grafts, which persisted long term with hair. There was a 2-fold increase in graft-infiltrating T cells in CD200-deficient skin at 14 d. Alopecia and skin lesions were induced in CD200-deficient hosts by adoptive transfer of splenocytes from WT mice previously grafted with CD200-negative skin, but not from mice grafted with WT skin. Collectively, these results suggest that the expression of CD200 in follicular epithelium attenuates inflammatory reactions and may play a role in maintaining immune tolerance to HF-associated autoantigens.

Although cellular immune responses are likely important for recovery from acute adenovirus infection, they have not been studied in humans. As a first step, a sensitive assay for the detection of adenovirus-specific proliferative T cell responses was developed. Peripheral blood mononuclear cells from 29 of 30 healthy adults exhibited specific proliferative responses to adenovirus type 2 antigen. Adenovirus-specific proliferation was found to be mediated by CD4+ T cells. Specific proliferative responses were detected to purified Ad2 virions and by Ad2-infected cell lysates, indicating that adenovirus structural proteins are important targets. In addition, specific proliferative responses to the uncommon adenovirus type 35 were found in persons without serologic evidence of prior Ad35 infection. These data suggest that adenovirus-specific CD4+ T cells recognize conserved antigens among different serotypes and that a majority of persons develop long-lived CD4+ T cell responses to adenovirus.

We describe a four-year experience with bone marrow transplantation involving closely HLA-matched unrelated donors and 55 consecutive patients with hematologic disease who were seven months to 48.6 years old (median, 18 years). An intensive pretransplantation conditioning regimen and graft-versus-host disease (GVHD) prophylaxis with CD3-directed T-cell depletion and cyclosporine were employed. Durable engraftment was achieved in 50 of 53 patients who could be evaluated (94 percent; 95 percent confidence interval, 83 to 98 percent). Acute GVHD of Grade II to IV developed in 46 percent of the patients (confidence interval, 27 to 66 percent). The incidence and severity of acute GVHD were increased in recipients of HLA-mismatched marrow as compared with recipients of phenotypically matched marrow (incidence of 53 percent [confidence interval, 37 to 68 percent] vs. 17 percent [confidence interval, 5 to 45 percent]; P less than 0.05). Extensive chronic GVHD and deaths not due to relapse also tended to be more frequent when HLA-mismatched marrow was used, but not significantly so. With a median follow-up of more than 19 months (range, greater than 9 to greater than 39), the actuarial disease-free survival of transplant recipients with leukemia and a relatively good prognosis (acute leukemia in first remission and chronic myelogenous leukemia in chronic phase) was 48 percent (confidence interval, 24 to 73 percent), and that of recipients with more aggressive leukemia was 32 percent (confidence interval, 18 to 51 percent); the actuarial survival of recipients with non-neoplastic disease was 63 percent (confidence interval, 31 to 86 percent). We conclude that marrow transplantation with closely HLA-matched unrelated donors can be effective treatment for neoplastic and non-neoplastic diseases. Although transplants from phenotypically HLA-matched unrelated donors appear to be most effective, transplants with limited HLA disparity can also be successful in some patients.