Regeneration and tolerance factor is expressed during T-lymphocyte activation and plays a role in apoptosis.

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.

Expression of regeneration and tolerance factor correlates directly with human immunodeficiency virus infection and inversely with hepatitis C virus infection.

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.

Regeneration and tolerance factor: a correlate of human immunodeficiency virus-associated T-cell activation.

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.

Increased expression of regeneration and tolerance factor in individuals with human immunodeficiency virus infection.

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.

Natural killer (NK) cell subsets and NK cell cytotoxicity in women with histories of recurrent spontaneous abortions.

PROBLEM: Natural Killer (NK) cell measurement and NK cytotoxicity are two measurements for assessing the cellular immune response. Both of the techniques have been reported to be prognostic for women with recurrent spontaneous abortion (RSA). We evaluated the two methods to determine the relationship of the two assays. Because both methods portend to evaluate the same process, the previous clinical data suggested that the methods evaluate the same phenomena. We undertook these studies to determine whether simple NK cell counts may be sufficient in the evaluation of NK activity in RSA. METHOD OF STUDY: The NK cell cytotoxicity at effector-to-target ratios of 50:1 and 25:1 was determined using a flow cytometric NK cell cytotoxicity assay. These values were then correlated with the percentages and absolute counts of three peripheral blood NK cell subsets. RESULTS: The data indicate that the flow cytometric assay is reproducible and precise and can be successfully used to evaluate patient samples. Linear regression analysis indicated a lack of correlation between peripheral blood NK cell cytotoxicity and percentages or absolute counts of CD56+CD16+, CD56+CD16- or CD3+CD56+ lymphocyte subsets (range of correlation coefficients, 0.1-0.3). CONCLUSIONS: NK cell cytotoxicity and peripheral blood NK cell values measure different aspects of NK cells and do not correlate. These data indicate that simple enumeration of NK cells may not be sufficient in the evaluation of NK cells in RSA.

Macrophages interact with enriched populations of distinct T lymphocyte subsets for the induction of severe destructive Lyme arthritis.

Severe destructive Lyme arthritis was detected in the hind paws of hamsters infused with enriched populations of either CD4+ or CD4- T lymphocytes along with macrophages exposed in vitro to formalin-inactivated Borrelia burgdorferi and then infected with the Lyme spirochete. Swelling was detected 4 days after infection, increased rapidly, peaked on day 8 of infection, and gradually decreased. Similarly, severe destructive arthritis was induced in hamsters infused with enriched populations of unfractionated T lymphocytes and macrophages exposed to spirochetes after infection with B. burgdorferi. Histopathological examination affirmed that hamsters infused with CD4+, CD4-, or unfractionated T lymphocytes and macrophages exposed to B. burgdorferi-induced arthritis. In addition, macrophages exposed in vitro to B. burgdorferi demonstrated both conventional and coiling phagocytosis, suggesting a mechanism by which CD4+ and CD4- T lymphocytes induce arthritis, respectively. These findings demonstrate that both CD4+ and CD4- subpopulations of T lymphocytes are capable of interacting with macrophages for the induction of severe destructive Lyme arthritis.

Macrophages and enriched populations of T lymphocytes interact synergistically for the induction of severe, destructive Lyme arthritis.

Hamsters receiving both macrophages exposed to Formalin-inactivated Borrelia burgdorferi (Mphi-FBb) and enriched populations of either immune or naive T lymphocytes developed severe swelling of the hind paws when infected with B. burgdorferi. Swelling was detected 6 days after infection, peaked on day 10, and gradually decreased. Swelling was also observed in the hind paws of hamsters infused with only Mphi-FBb or only enriched populations of either immune or naive T lymphocytes after infection with B. burgdorferi. However, the swelling detected in these hamsters was less severe and of shorter duration. In addition, hamsters receiving both macrophages not exposed to Formalin-inactivated B. burgdorferi (Mphi-NFBb) and enriched populations of either immune or naive T lymphocytes failed to develop severe swelling after infection with B. burgdorferi. No swelling was also observed in hamsters infused with both Mphi-FBb and enriched populations of immune T lymphocytes and then inoculated with spirochetal growth medium. We further showed that macrophages and enriched populations of T lymphocytes did not interact synergistically for controlling B. burgdorferi infection, as spirochetes were readily recovered from the tissues of all cell transfer recipients infected with B. burgdorferi. These findings demonstrate that hamsters infused with both Mphi-FBb and enriched populations of either immune or naive T lymphocytes develop a more fulminate arthritis after infection with B. burgdorferi than recipients infused with either cell type alone. These findings suggest that macrophages and T lymphocytes interact synergistically for the induction of severe, destructive Lyme arthritis.

Characterization of the protective borreliacidal antibody response in humans and hamsters after vaccination with a Borrelia burgdorferi outer surface protein A vaccine.

Significant borreliacidal antibody was induced in volunteers and hamsters 60 days after primary and secondary vaccination with high concentrations of recombinant outer surface protein A (rOspA). However, the borreliacidal antibody response waned rapidly. Only 1 person had detectable cidal activity 180 days after vaccination. Similarly, the borreliacidal antibody response waned rapidly in hamsters by week 10 of vaccination. By contrast, the total anti-rOspA antibody response remained elevated in volunteers and hamsters. When isolates of Borrelia burgdorferi sensu lato were incubated in sera from vaccinated humans or hamsters, only the vaccine-specific isolate was killed. These results were confirmed by challenging rOspA-vaccinated hamsters with different isolates of B. burgdorferi sensu lato. The results showed that monitoring total rOspA antibody is inappropriate for evaluating the efficacy of an rOspA vaccine. The rOspA vaccine must be improved to yield comprehensive protection and maintain sustained levels of protective borreliacidal antibodies.

Involvement of CD4+ T lymphocytes in induction of severe destructive Lyme arthritis in inbred LSH hamsters.

We determined that Borrelia burgdorferi-specific CD4+ T lymphocytes are responsible for the development of severe destructive Lyme arthritis and affect the production of borreliacidal antibody. Severe destructive Lyme arthritis was readily evoked in immunocompetent inbred LSH hamsters vaccinated with a whole-cell preparation of Formalin-inactivated B. burgdorferi sensu stricto isolate C-1-11 in adjuvant when challenged with B. burgdorferi sensu stricto isolate 297. When vaccinated hamsters were depleted of CD4+ T lymphocytes by the administration of monoclonal antibody GK1.5 and challenged, they failed to develop severe destructive arthritis. Similarly, nonvaccinated hamsters with or without the depletion of CD4+ T lymphocytes failed to develop severe destructive arthritis. In addition, depleting CD4+ T lymphocytes impaired the development of borreliacidal antibody in vaccinated and nonvaccinated hamsters challenged with the Lyme borreliosis spirochete. These findings show that CD4+ T lymphocytes are important for the recognition of arthritogenic and protective antigens of B. burgdorferi sensu lato isolates. Additional studies are needed to define the mechanisms responsible for the development of severe destructive Lyme arthritis and the production of borreliacidal antibody.

Abilities of OspA proteins from different seroprotective groups of Borrelia burgdorferi to protect hamsters from infection.

The ability of vaccination with recombinant OspA from six seroprotective groups of Borrelia burgdorferi sensu lato to induce protection against infection with homologous and other Lyme spirochetes was examined in hamsters. Antisera generated against the OspA proteins of B. burgdorferi sensu stricto S-1-10 and C-1-11 (seroprotective groups 1 and 2, respectively), Borrelia afzelii BV1 (seroprotective group 4), and Borrelia garinii LV4 (seroprotective group 5) were able to kill the homologous spirochete in vitro but not other isolates. Surprisingly, antisera against B. afzelii PKo (seroprotective group 6) and B. burgdorferi sensu lato LV5 (seroprotective group 3) OspA proteins were unable to kill the homologous organism, although LV5 OspA antisera killed the heterologous isolates S-1-10 and LV4. In vivo vaccination studies supported the in vitro findings, confirming that vaccination with a single OspA protein does not provide complete protection against challenge with all Lyme disease spirochetes. In addition, OspA antibodies from some isolates may not protect against the homologous isolate. The induction of protective antibodies against other B. burgdorferi proteins may be necessary to insure a comprehensive Lyme disease vaccine.

Borrelia burgdorferi-specific T lymphocytes induce severe destructive Lyme arthritis.

This is the first documentation that Borrelia burgdorferi-specific T lymphocytes are involved in the pathogenesis of Lyme arthritis. We present direct evidence that T lymphocytes obtained from inbred LSH hamsters vaccinated with a whole-cell preparation of Formalin-inactivated B. burgdorferi sensu stricto isolate C-1-11 in adjuvant conferred on naive recipient hamsters the ability to develop severe destructive arthritis when challenged with B. burgdorferi sensu stricto isolates C-1-11 and 297. By contrast, recipients infused with normal T lymphocytes and challenged with B. burgdorferi sensu stricto isolates C-1-11 and 297 failed to develop severe destructive arthritis. The T lymphocytes transferred were obtained from the lymph nodes of vaccinated and nonvaccinated hamsters by depleting B lymphocytes by using monoclonal antibody 14-4-4s (< 1% B lymphocytes by flow cytometric analysis). The enriched T lymphocytes showed enhanced proliferation to stimulation with concanavalin A and failed to respond to lipopolysaccharide. Moreover, only the enriched T lymphocytes from vaccinated hamsters proliferated on exposure to a whole-cell preparation of B. burgdorferi sensu stricto isolate C-1-11 in the presence of mitomycin-treated syngeneic antigen-presenting cells. These results demonstrate that B. burgdorferi-specific T lymphocytes primed by vaccination with a whole-cell preparation of inactivated B. burgdorferi sensu stricto isolate C-1-11 in adjuvant are involved in the development of severe destructive arthritis. Additional experiments are needed to define the precise mechanism(s) responsible for the development of Lyme arthritis.

Development of destructive arthritis in vaccinated hamsters challenged with Borrelia burgdorferi.

We present the first direct evidence that adverse effects, particularly severe destructive arthritis, can develop in vaccinated hamsters after challenge with Borrelia burgdorferi sensu lato isolates. Hamsters were vaccinated with a whole-cell preparation of Formalin-inactivated B. burgdorferi sensu stricto isolate C-1-11 in adjuvant. A severe destructive arthritis was readily evoked in vaccinated hamsters challenged with the homologous B. burgdorferi sensu stricto isolate C-1-11 before high levels of protective borreliacidal antibody developed. Once high levels of C-1-11 borreliacidal antibody developed, hamsters were protected from homologous challenge and development of arthritis. Vaccinated hamsters, however, still developed severe destructive arthritis when challenged with other isolates of the three genomic groups of B. burgdorferi sensu lato (B. burgdorferi sensu stricto isolate 297, Borrelia garinii isolate LV4, and Borrelia afzelii isolate BV1) despite high levels of C-1-11 specific borreliacidal antibody. Vaccines that contained whole spirochetes in adjuvant induced destructive arthritis, but this effect was not dependent on the isolate of B. burgdorferi sensu lato or the type of adjuvant. These studies demonstrate that caution is necessary when employing whole spirochetes in adjuvant for vaccination to prevent Lyme borreliosis. Additional studies are needed to identify the antigen(s) responsible for the induction and activation of arthritis and to define the immune mechanisms involved.

Seroprotective groups of Lyme borreliosis spirochetes from North America and Europe.

Five distinct seroprotective groups among North American and European isolates of Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii have been identified using the in vitro borreliacidal assay. The predominant North American seroprotective group comprised isolate 297 and B. burgdorferi isolates from California, Illinois, Wisconsin, New York, and Texas. A second group was represented by isolate C-1-11. The majority of European isolates belonged to a seroprotective group composed of B. garinii. Another European group contained isolates classified genetically as genospecies group VS461 (B. afzelii). A fifth group, represented by isolate LV5, could kill both North American and European isolates of B. burgdorferi sensu lato. These results suggest that combinations of immunogenic protective proteins of spirochetes will be necessary to provide a comprehensive vaccine.