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Regeneration and tolerance factor (RTF) is a protein with immunosuppressive activity and is normally present in the thymus and placenta. RTF was measured in the livers of patients with regenerating nodules due to alcoholic cirrhosis and hepatitis C. RTF was expressed in the regenerating nodules of 26 patients with alcoholic cirrhosis. All patients with chronic hepatitis C without cirrhosis failed to express RTF. Flow cytometry revealed upregulation of RTF on the lymphocytes from alcoholic cirrhosis and downregulation in hepatitis C disease.

Regeneration and tolerance factor (RTF) is a protein cloned from the thymus and expressed on B lymphocytes in normal pregnancy, B lymphocytic leukemia lines, and T and B lymphocytes in individuals with HIV infection. Findings, using the Jurkat T-cell model, revealed that RTF is upregulated after activation and anti-RTF antibody-induced apoptosis. In this article anti-RTF antibody-induced apoptosis of both unstimulated and activated T lymphocytes. RTF expression was examined in human PBMC or purified T lymphocytes after their in vitro activation. Kinetic studies indicated maximal RTF cell surface expression on activated T lymphocytes occurred between expression of the early activation antigen CD69 and the IL-2alpha receptor (CD25) by multiparameter flow cytometry. RTF receptor expression correlated with Fas (CD95) and CD25 receptor expression (r2 = 0.6 and 0.5, respectively). RTF surface expression was dependent on the stimuli used to activate T lymphocytes. T lymphocytes obtained maximal RTF expression when activated through the TCR signal complex using anti-CD3epsilon antibody alone when compared with T lymphocytes activated with costimulation provided by anti-CD28 antibody alone or with anti-CD28 and anti-CD3epsilon antibody. RTF is expressed under conditions of both activation and anergy. The RTFs increased concentration on the surface of anergic T cells may protect these cells from apoptosis because increased RTF concentrations inhibited anti-RTF induced apoptosis. These data further characterize the expression of RTF on activated T lymphocytes and the role of anti-RTF antibody in T-lymphocyte apoptosis.

Regeneration and tolerance factor (RTF) was originally identified in the placenta of mice and the isolated protein shown to have suppressive effects. In these studies, the gene cloned from thymus tissue was mapped to human chromosome 12. The role of recombinant RTF on cytokines was examined. In addition, we examined the human placenta by immunohistochemistry for RTF expression. RTF was expressed at the peripheral layer of cytotrophoblast in 7-9-week-old placentas. Using the RTF gene sequence, a recombinant protein was prepared and shown to induce IL-10 production. These data indicate that RTF is expressed by the tissues most intimately involved at the maternal-fetal interface, and its biological activity is capable of producing the necessary immune response for initiating and maintaining the maternal-fetal relationship.

Regeneration and tolerance factor (RTF) is a novel membrane protein that has a diverse expression pattern and immunoregulatory properties. RTF is expressed in vivo on the surface of individuals with B cell chronic lymphocytic leukemia and on activated T lymphocytes of HIV infected individuals as determined by their coexpression with CD38 and HLA-DR. The unique expression patterns of this protein in vivo lead us to investigate its expression in vitro. The activation of human PBMCs through the TCR, using anti-CD3 antibody and PMA, upregulated cell surface expression of RTF from 2. 3% to 91.2% (mean channel fluorescence [MCF] increased threefold). The activation of Jurkat T cells through the TCR upregulated surface expression of RTF from 8.3% (MCF-1.3) to 58.7% (MCF-13.1). The Jurkat T-cell line was used as a model system to explore RTF's role in cellular activation. Using the Jurkat T-cell model, we found anti-RTF antibody induces apoptosis. The addition of anti-RTF antibody increased annexin V binding by threefold compared with the IgG1 kappa isotype control antibody (p < 0.00002) and activated caspase 3. These data indicate that RTF is expressed during T-cell activation and may be associated with apoptosis.

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) cause two of the most prevalent debilitating viral infections. HIV appears to induce a skewing toward a Th2 response, while in HCV infection a Th1 response appears to dominate. Regeneration and tolerance factor (RTF) may participate in driving or sustaining a Th2 cytokine response. The expression of RTF on CD3(+) T cells of HIV-seropositive (HIV(+)) individuals is increased. The purpose of this study was to compare the expression of RTF during HIV infections with that during HCV infections. Three-color flow-cytometric analysis of peripheral blood collected from HIV(+) HCV-seropositive (HCV(+)), HIV- and HCV-seropositive (HIV(+) HCV(+)), and HIV- and HCV-seronegative (HIV(-) HCV(-)) individuals was performed. Levels of RTF expression on T-lymphocyte subsets from these groups were compared, as were levels of RTF expression on activated T cells expressing CD38 and HLA-DR, to determine the relationship of RTF expression to these infections. We demonstrated that the expression of RTF on surfaces of T cells from HIV(+) individuals is upregulated and that its expression on T cells from HCV(+) individuals is downregulated. A twofold increase in the mean channel fluorescence of RTF on CD3(+) T cells was seen in both HIV(+) and HIV(+) HCV(+) individuals compared to HIV(-) HCV(-) individuals. HCV(+) individuals had lower levels of RTF expression than HIV(-) HCV(-) individuals (P < 0.005 for CD4(+); P < 0.0005 for CD8(+)). In terms of percentages of T cells expressing RTF, the groups were ranked as follows: HIV(+) > HIV(+) HCV(+) > HIV(-) HCV(-) > HCV(+). The results indicate that RTF expression correlates with HIV-associated immune activation and may be associated with Th2-type responses.

Human immunodeficiency virus (HIV) infection causes extensive phenotypic alterations in lymphocytes. Cellular markers that are normally absent or expressed at low levels on quiescent cells are upregulated throughout the disease course. The transmembrane form of regeneration and tolerance factor (RTF) is expressed at negligible levels on resting T cells but is quickly upregulated following in vitro stimulation and activation. Recently, we reported that expression of RTF was significantly higher in cells from HIV-seropositive (HIV(+)) individuals than in cells from HIV-seronegative (HIV(-)) individuals. Because T cells from HIV(+) individuals express markers reflecting chronic activation, we hypothesized that these in vivo-activated cells would coexpress RTF. Flow cytometry was used to assess RTF expression on activated (CD38(+) and HLA-DR(+)) CD4(+) and CD8(+) T cells. HIV(+) individuals had higher percentages of RTF(+) CD38(+) (P < 0.0001) or RTF(+) HLA-DR(+) (P = 0.0001) CD4(+) T cells than HIV(-) individuals. In HIV(+) individuals, increased percentages of CD4(+) T cells that were RTF(+), RTF(+) CD38(+), and RTF(+) HLA-DR(+) correlated inversely with the absolute number and percentage of CD4(+) T cells and correlated positively with plasma beta(2)-microglobulin concentrations. HIV(+) individuals had higher percentages of CD8(+) T cells that were RTF(+) CD38(+) (P = 0.0001) or RTF(+) HLA-DR(+) (P = 0.0010). In HIV(+) individuals, increased percentages of CD8(+) T cells that were RTF(+) HLA-DR(+) correlated inversely with the percentage of CD4(+) T cells, and high percentages of CD8(+) T cells that were RTF(+) CD38(+) correlated positively with plasma beta(2)-microglobulin levels. These findings strongly suggest that increased RTF expression is a correlate of HIV-associated immune system activation.

Regeneration and tolerance factor (RTF) plays a pivotal role in successful pregnancy outcome and has potent immunomodulating properties. During pregnancy, it is abundantly expressed in the placenta and on peripheral B lymphocytes. Several lines of evidence suggest that both successful pregnancy outcome and progression from human immunodeficiency virus (HIV) infection to AIDS are associated with a Th2-type response. As a result, we hypothesized that the cellular expression of RTF may also be increased during infection with HIV. Using flow cytometric analysis, we showed a significantly (P < 0.01) increased expression of RTF on CD3(+) cells obtained from individuals with HIV over that for individuals without HIV. On average, 32.1% of the CD3(+) cells from individuals with HIV expressed high levels of RTF. In contrast, an average of only 6.7% of the CD3(+) cells from individuals without HIV expressed high levels of RTF. Similar results were obtained when CD19(+) cells from individuals with (mean, 44.1%) and without (mean, 25.8%) HIV were evaluated. Linear regression analysis suggested that high levels of RTF expression by CD3(+) cells correlated better with viral load (r value, 0.46) than with absolute CD4 count (r value, 0.09). While additional experiments are necessary to delineate the precise immunologic role of RTF, our current data suggest that RTF expression during HIV infection may be a useful marker of immune activation.

Problem: TJ6 will be one of the molecules involved in fetal-specific immune suppression during pregnancy. In the mouse and human decidua, the regulation of uterine natural killer (uNK) cells is important during pregnancy., Method of Study: To further understand the possible functions of TJ6 during pregnancy, syngeneic, allogeneic, and mutant mice were examined for TJ6 expression., Results: Immunoblotting showed that TJ6 protein was expressed on most of the placenta-associated mononuclear cells, and the size was 70-72 kDa at all stages of pregnancy. The expression of TJ6 mRNA was studied by a ribonuclease protection assay in syngeneic and allogeneic matings, and in immune-deficient mice of genotypes scid/scid and scid/scid.bg/bg., Conclusions: Genetic disparity, lack of T and B lymphocytes, and loss of NK lytic function had no significant effect on the expression of TJ6 mRNA.

TJ6, a newly described protein produced locally in the uterine decidua during pregnancy, may be involved in maintaining a unique immunological environment at the maternal-fetal interface. The aim of this study was to determine whether TJ6 is expressed as membrane form on decidual lymphocytes (DL), to define the phenotypes of TJ6m (membrane form TJ6) expressing cells and to analyze the fluorescence intensity of TJ6m expression. Peripheral blood lymphocytes (PBL) and DL were obtained from first trimester pregnancies undergoing elective termination and immunophenotyped for TJ6m and other cell surface antigens (CD3, CD8, CD19, CD56, CD16) by flow cytometry. This is the first study showing that TJ6 molecules are present on decidual lymphocytes in human pregnancy. TJ6m expression on PBL was not different from that of DL. However, a significantly higher percentage of double positive (TJ6m+CD3+, TJ6m+,CD8+,TJ6m+CD19+) cells were found in PBL when compared to DL. The average fluorescence intensity (AFI) for the TJ6m marker among cells with CD8+, CD19+ and CD56+ double positive was significantly higher in DL as compared with those of PBL. The AFI for granularity of double positive DL was significantly higher than observed in PBL.

Problem: TJ6 is a cytokine whose membrane form is regularly expressed on the B lymphocytes of women during pregnancy. In a previous study we have shown that pregnancies that end in a spontaneous abortion are characterized by an increase in natural killer (NK) cells and that NK cells in these pregnancies also expressed TJ6, whereas NK cells from pregnancies that terminate normally neither increase in number nor do they express significant amounts of the cytokine., Methods: To evaluate the ability of TJ6 to predict pregnancy outcome, 61 blood samples from pregnant women were studied. Blood samples were drawn between 5 and 12 weeks of gestation and analyzed for lymphocyte expression of TJ6 using Cytoron Absolute flow cytometry and two-color fluorescence. The percentage of CD19+ (B) cells and CD56+ (NK) cells that expressed TJ6 was calculated, and this percentage was correlated with subsequent pregnancy outcome classified as successful (viable birth) or unsuccessful (abortion, stillbirth). TJ6 CD19 and TJ6 CD56 was determined in 32 blood samples from women with successful pregnancy outcomes and 29 samples from women with unsuccessful pregnancy outcome., Results: The mean TJ6 CD19 expression for successful pregnancies was 10.6% and for unsuccessful pregnancies was 5.1% (P < 0.03). The mean TJ6 CD56+ percentage of circulating cells that express TJ6 expression for successful pregnancies was 3.3% and for unsuccessful pregnancies was 10.4% (P = 0.02). All unsuccessful pregnancies had less than 90% of circulating CD19 cells express TJ6 and/or greater than 50% of circulating CD56 cells express TJ6. Use of the expression of TJ6 on CD19 and CD56 cells during the first trimester predicts viable pregnancy with a sensitivity of 100%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 100%., Conclusions: The work now being reported validates the hypothesis that expression of TJ6 on NK cells obtained from pregnant women predicts the outcome of pregnancy. In addition we show that this prognosticator can be reliably demonstrated early in the first trimester.

Problem: Characterization of the soluble form of a novel protein, TJ6 (TJ6s) with immune suppressive activity from murine fetoplacental units., Method: Preferential ammonium sulfate precipitation, gel filtration and ion-exchange chromatography were employed to purify the protein TJ6s from murine fetoplacental units using an anti-peptide antibody as a detection tool. Biological activity of the purified protein was studied in lymphocyte proliferation assays., Results: Purified TJ6s has a M(r) of approximately 18 kDa as evidenced by SDS-PAGE in both reducing and non reducing conditions. It exerted a strong anti-proliferative activity in both anti-CD3 and Con A proliferation lymphocyte proliferation assays but not in a PHA assay, suggesting that the anti-proliferative effects on T cells are exerted only on cells specifically activated directly through T cell receptor complex., Conclusion: The results indicate that TJ6s is a novel anti-proliferative protein that has many of the characteristics that are considered necessary for survival of the fetal allograft.

TJ6 is a novel protein which has immunosuppressive activity and may have a functional role in fetal allograft survival during pregnancy. Initial studies indicated that when mice were treated with an anti-TJ6 binding mAb early in pregnancy, the pregnancies were completely ablated and that TJ6 expression is enhanced dramatically during pregnancy. In addition we have cloned the cDNA for TJ6 which encodes a possible transmembrane domain that may include six to seven transmembrane regions. Therefore, we examined TJ6 expression on PBL of pregnant and non-pregnant women and found that TJ6 is expressed primarily on CD19+ B cells from pregnant but not nonpregnant women. TJ6 was not expressed on CD3+ lymphocytes from either group but was expressed on CD56+ cells from a small population of pregnant women which preliminary data indicate may correlate with the occurrence of spontaneous abortion in these women. Here we also show that TJ6 transcripts are highly expressed in the developing fetoplacental unit as well as in the developing thymus. We also begin to characterize the expression of TJ6 isoforms in an acute lymphocytic leukemia cell line (SB), murine thymus, and the developing murine fetoplacental unit, as well as the expression of a membrane form of TJ6 present on human lymphocytes during pregnancy. All of these cells and tissues expressed TJ6 proteins which were smaller than predicted based on either the cDNA sequence or the in vitro translation even though they expressed mRNA similar in size. The TJ6 isoforms varied in size from the 45-kDa isoform in SB cells to the 52-kDa isoform of the fetoplacental unit to a 70-kDa isoform in murine thymus. Flow cytometric analysis also demonstrated that similar to the CD19+ B cells from pregnant women, TJ6 is expressed on the surface of SB cells.