Therapeutic potential of CD8+ cytotoxic T lymphocytes in SLE.

Recent evidence supports the idea that following a break in tolerance, CD8 cytotoxic T lymphocytes (CTL) may be an important but unrecognized mechanism for limiting expansion of autoreactive B cells. Failure of this mechanism could allow persistence of CD4 T cell driven polyclonal B cell activation resulting in clinical lupus. Although CD8 CTL failure may occur early in disease, work in mice supports the concept that therapeutic CTL enhancement may be both practical and beneficial in lupus. Devising such therapy for humans will first require an understanding of the in vivo mechanisms critical in CTL expansion and down regulation, particularly in the lupus setting which may differ from CTL generation in other clinical settings (e.g. tumors, infections).

Active-edge planar radiation sensors.

Many systems in medicine, biology, high-energy physics, and astrophysics require large area radiation sensors. In most of these applications, minimizing the amount of dead area or dead material is crucial. We have developed a new type of silicon radiation sensor in which the device is active to within a few microns of the mechanical edge. Their perimeter is made by a plasma etcher rather than a diamond saw. Their edges can be defined and also passivated by growing, in an intermediate step, a field oxide on the side surfaces. In this paper, the basic architecture and results from a synchrotron beam test are presented.

The Parent-into-F1 Model of Graft-vs-Host Disease as a Model of In Vivo T Cell Function and Immunomodulation.

Since its description roughly 30 years ago, the parent-into-F1 model of graft-vs.-host disease has provided insights into the mechanisms of in vivo T cell activation and the pathogenesis of autoimmune conditions. A new and emerging role for the P-->F1 model is one of identifying agents with immunomodulatory activity and defining in vivo mechanisms that promote cell mediated or antibody mediated immune responses. Because F1 mice are not irradiated prior to donor cell transfer, the P-->F1 model has in the past not been strictly analogous to human hematopoetic stem cell transplantation. However with the advent of newer non-myeloablative conditioning regimens, the model may assume more relevance. In this article, we first provide a review of relevant earlier fundamental observations followed by a summary of recent work from our laboratory in which acute and chronic GVHD in this model have been used not only to study normal T cell responses in vivo but also to define mechanisms important in the pathogenesis of autoimmunity and immunomodulation.

In vivo neutralization of TNF-alpha promotes humoral autoimmunity by preventing the induction of CTL.

Neutralization of TNF-alpha in humans with rheumatoid arthritis or Crohn's disease has been associated with the development of humoral autoimmunity. To determine the effect of TNF-alpha neutralization on cell-mediated and humoral-mediated responses, we administered anti-TNF-alpha mAb to mice undergoing acute graft-vs-host disease (GVHD) using the parent-into-F(1) model. In vivo neutralization of TNF-alpha blocked the lymphocytopenic features characteristic of acute GVHD and induced a lupus-like chronic GVHD phenotype (lymphoproliferation and autoantibody production). These effects resulted from complete inhibition of detectable antihost CTL activity and required the presence of anti-TNF-alpha mAb for the first 4 days after parental cell transfer, indicating that TNF-alpha plays a critical role in the induction of CTL. Moreover, an in vivo blockade of TNF-alpha preferentially inhibited the production of IFN-gamma and blocked IFN-gamma-dependent up-regulation of Fas; however, cytokines such as IL-10, IL-6, or IL-4 were not inhibited. These results suggest that a therapeutic TNF-alpha blockade may promote humoral autoimmunity by selectively inhibiting the induction of a CTL response that would normally suppress autoreactive B cells.

Role of perforin in controlling B-cell hyperactivity and humoral autoimmunity.

To determine the role of perforin-mediated cytotoxic T lymphocyte (CTL) effector function in immune regulation, we studied a well-characterized mouse model of graft-versus-host disease (GVHD). Induction of acute GVHD using perforin-deficient donor T cells (pfp-->F1) initially resulted in features of acute GVHD, e.g., engraftment of both donor CD4(+) and CD8(+) T cells, upregulation of Fas and FasL, production of antihost CTL, and secretion of both Th1 and Th2 cytokines. Despite fully functional FasL activity, pfp donor cells failed to totally eliminate host B cells, and, by 4 weeks of disease, cytokine production in pfp-->F1 mice had polarized to a Th2 response. Pfp-->F1 mice eventually developed features of chronic GVHD, such as increased numbers of B cells, persistence of donor CD4 T cells, autoantibody production, and lupuslike renal disease. We conclude that in the setting of B- and T-cell activation, perforin plays an important immunoregulatory role in the prevention of humoral autoimmunity through the elimination of both autoreactive B cells and ag-specific T cells. Moreover, an ineffective initial CTL response can evolve into a persistent antibody-mediated response and, with it, the potential for sustained humoral autoimmunity.

Differential expression of Fas and Fas ligand in acute and chronic graft-versus-host disease: up-regulation of Fas and Fas ligand requires CD8+ T cell activation and IFN-gamma production.

The parent-into-F1 model of acute and chronic graft-vs-host disease (GVHD) was used as an example of in vivo cell-mediated or Ab-mediated responses, respectively, and the roles of Fas and Fas ligand (FasL) were investigated. Using both flow cytometry and PCR methodologies, we found that acute GVHD mice exhibited significant up-regulation of Fas and FasL, whereas Fas/FasL up-regulation in chronic GVHD mice was equal to or marginally greater than that in uninjected mice. Functional studies confirmed that Fas/FasL contributed to the anti-host CTL activity of splenocytes from acute GVHD mice, although a perforin-dependent pathway was also identified. Despite the presence of FasL on both donor CD4+ and CD8+ T cells in acute GVHD mice, depletion studies demonstrated that all the in vitro anti-host CTL activity resided in the CD8+ population. Furthermore, injection of CD8-depleted B6 spleen cells into F1 mice blocked Fas/FasL up-regulation and IFN-gamma production, resulting in chronic GVHD. Lastly, up-regulation of Fas/FasL in acute GVHD mice could be blocked by anti-IFN-gamma mAb in vivo. Thus, in this in vivo model of alloantigen immune responsiveness, Fas/FasL up-regulation is critically dependent on Ag-specific (donor) CD8+ T cell activation and IFN-gamma production. Donor CD4+ T cell activation in the absence of CD8+ T cell activation results in an autoantibody-mediated response, no significant Fas/FasL up-regulation, impaired elimination of autoreactive B cells, and persistent humoral autoimmunity.

Genetic analysis of experimentally induced lupus in mice.

The DBA/2 and C57BL/6 mouse strains, as well as the BXD RI lines derived from these strains, were used to map the genes controlling experimentally induced systemic lupus erythematosus (SLE). SLE was induced using two immunologic approaches: (1) immunization with the human monoclonal anti-DNA antibody expressing the 16/6Id, to which the DBA/2 strain is susceptible (responder) and the C57BL/6 strain is resistant (nonresponder); and (2) induction of autoimmune GVHD in B6D2F1 hosts by inoculation of parental DBA/2 (induces SLE) or C57BL/6 (does not induce SLE) T cells. By both approaches the BXD RI lines could be divided into distinct DBA/2-like and C57BL/6-like categories. Concordance of SLE induced by both methods was observed for susceptibility and resistance in 13/15 BXD lines (P < 0.005). The results suggest that at least two non-H-2 genes control susceptibility and resistance to experimentally induced SLE, one mapping to chromosome 7 and the other mapping to chromosome 14.

The epithelial cell-specific integrin, CD103 (alpha E integrin), defines a novel subset of alloreactive CD8+ CTL.

The interaction of CD8+CTL with epithelial layers is an important but poorly defined aspect of organ allograft rejection. We herein report that CD103 (formerly alpha E integrin), a known receptor for the epithelial cell-specific ligand E-cadherin, is expressed by a major subset of CD8 + CTL elicited in response to allogeneic renal epithelial cells (REC). In contrast, CD103 was expressed poorly on CD8 + CTL generated in the conventional manner by stimulation with allogeneic leukocytes, although expression could be dramatically up-regulated by supplementing cultures with REC or exogenous TGF-beta 1. That TGF-beta controls the expression of CD103 on CD8+ CTL was further supported by the capacity of anti-TGF-beta mAb to block the generation of such cells in anti-REC cultures. Clonal analyses of anti-REC cultures revealed that individual CD8+ CTL clones were discretely CD103+ or CD103-, nd maintained their respective phenotypes independently of the cell type used for clonal restimulation. In a mouse model of graft-vs-host disease, 16.4 +/- 2.7% of CD8 cells that infiltrated host kidneys were CD103+ (n = 4). CD8 kidney-infiltrating lymphocytes were predominantly of donor origin and displayed an activated/memory phenotype (CD62L-, CD44high), consistent with expression of CD103 on a CD8 effector subset elicited in vivo following allogeneic transplantation. Taken together, the present data demonstrate that CD103 identifies a novel CD8 effector subset and, moreover, that such cells may comprise a significant component of the response to allogeneic tissues. The potential for CD103+ CTL as an important effector mechanism in organ allograft rejection, and more generally, as a mechanistic basis for tissue-specific immune phenomena, is discussed.

Do effector and memory T helper cells also need B7 ligand costimulatory signals?

Blocking B7 ligand-costimulatory molecules can inhibit a primary T-dependent immune response, but whether these interactions also mediate ongoing or memory immune responses is less clear. Development of immunotherapies based on blocking B7 ligand interactions would be limited if they were effective only at the initiation of an immune response. We discuss the conditions under which T helper effector and memory cells may or may not require B7 ligand interactions for their function.

A major role for the Fas pathway in acute graft-versus-host disease.

Recent studies have suggested a role for the Fas pathway in the wasting syndrome associated with lpr-->wild-type bone marrow transplants. To directly examine whether Fas ligand has a major role in the development of acute graft-vs-host disease (GVHD), Fas ligand-deficient (gld) mice were used as donors and C3H/HeJ x C57BL/6F1 as recipients in the parent-into-F1 model of acute GVHD. Transplantation of C3H/gld spleen cells induced significantly less host lymphoid depletion and was associated with less antihost cytotoxic activity in vitro when compared with wild-type C3H donor cells. The reduced depletion of host lymphocytes was explained by both impaired antihost T cell cytolytic activity and by reduced expansion of gld donor T cells in F1 recipients. These findings not only indicate that the Fas ligand is an important effector molecule in acute GVHD, but also provide in vivo evidence supporting a role for Fas/Fas ligand interactions in T cell expansion and maturation.

Differential effect of CTLA4Ig on murine graft-versus-host disease (GVHD) development: CTLA4Ig prevents both acute and chronic GVHD development but reverses only chronic GVHD.

The role of costimulation was examined in an in vivo model of alloantigen-driven Th1 or Th2 cytokine responses, the parent-into-F1 model of acute or chronic graft-vs-host disease (GVHD), respectively. The soluble fusion protein, murine CTLA4Ig, which blocks engagement of CD28 by its natural ligand B7-1 and B7-2, was administered either early, at the time of GVHD induction, or delayed, after the establishment of Th1 or Th2 effector responses (day 7). Early administration of CTLA4Ig prevented the development of both acute and chronic GVHD by preventing the activation of donor T cells, i.e., by blocking characteristic Th1 or Th2 cytokine production and blocking memory marker up-regulation on donor T cells. Delayed CTLA4Ig administration was unable to alter acute GVHD but did reverse chronic GVHD as evidenced by normalization of serum autoantibody levels, normal host B cell numbers and MHC class II expression, reduced donor T cell expression of CD40 ligand, and reduced numbers of donor CD4+ memory T cells. The percentage of donor memory cells was not altered by delayed CTLA4Ig. We conclude that in this model, alloantigen-driven Th1 or Th2 responses are equally susceptible to costimulatory blockade at the onset of disease; however, once effector mechanisms become established, only Th2-driven responses have a requirement for further costimulation for the continued expansion of CD4+ T cells. These data suggest that humoral, lupus-like autoimmunity requires continuous T cell help for B cells, and agents that interrupt this process may be beneficial.

Defective antigen-presenting cell function in patients with systemic lupus erythematosus.

OBJECTIVE: Patients with systemic lupus erythematosus (SLE) who exhibit defective in vitro responses to recall antigens and normal responses to alloantigens have been shown to have an abnormality in antigen-presenting cell (APC) function. This study was undertaken to further characterize this defect in APC function in lupus patients. METHODS: Mononuclear cells (MNC) from the peripheral blood of patients with SLE and from normal individuals were cultured in the presence of either recall antigen tetanus toxoid (TT), anti-CD3 (OKT3) monoclonal antibody, or alloantigens, and proliferative or interleukin-2 responses were assessed. Cell surface expression of B7-1 was assessed by flow cytometry. RESULTS: MNC from all normal individuals and from 7 patients with SLE responded to both TT and alloantigen and were designated +/+. Twelve SLE patients did not respond to TT but did respond to alloantigen stimulation and were designated -/+. In both normal subjects and SLE patients, the ability to respond to OKT3 correlated strongly with the ability to respond to recall antigen. A defect in APC costimulatory function was suggested by data demonstrating that interferon-gamma-induced expression of B7-1 was significantly reduced in SLE patients compared with controls. Neither controls nor SLE patients expressed detectable amounts of surface B7-1 molecule on resting APC. Defective recall and anti-CD3-stimulated responses could be enhanced in SLE patients in the presence of B7/BBl-transfected P815 murine mastocytoma cells underscoring an SLE-associated defect in costimulatory activity. However, nontransfected P815 cells were also able to enhance responses to OKT3 in -/+ patients; blocking experiments showed that this was mediated through an IgG Fc receptor-dependent mechanism. CONCLUSION: These data indicate that SLE-associated defects in APC function in vitro can be accounted for by abnormalities in APC surface membrane molecules such as B7, IgG Fc receptors, and possibly others.

Enhancement of BALB/c 3T3 cells transformation by 1,2-dibromoethane promoting effect.

Two of the most representative halogenated aliphatic hydrocarbons, 1,2-dibromoethane and 1,1,2,2-tetrachloroethane, were tested in the two-stage cell transformation model for analysing the promoting ability. Both of these compounds had previously been found to exert genotoxic effects, probably acting as moderate initiators. BALB/c 3T3 cells were initiated with subtransforming doses of N-methyl-N-nitro-N-nitrosoguanidine or 3-methylcholanthrene and then exposed to a chronic treatment with different non-transforming dosages of the two haloalkanes. 1,1,2,2-Tetrachloroethane did not exert any promoting activity in that system. By contrast, significant promoting effects by 1,2-dibromoethane were observed both in cells treated with N-methyl-N-nitro-N-nitrosoguanidine and in cells treated with 3-methylcholanthrene. Promotion of the transformation process initiated with 3-methylcholanthrene was detectable when confluent cells in the chemical-treated plates were replated in the level-II amplification test. This experimental procedure allowed cells to perform further rounds of replications and transformed foci to became detectable. Results gave evidence for a promoting role of 1,2-dibromoethane in multistep carcinogenesis, probably responsible for the higher oncogenic ability of this compound with respect to 1,1,2,2-tetrachloroethane.

Preliminary Studies of in vitro and in vivo Effects of Misoprostol on Th-1 and Th-2 Cytokine Production.

Prostaglandins of the E series are known to suppress in vitro production of Th-1 cytokines such as interleukin-2 (IL-2) and interferon-gamma but have not been shown to suppress production of Th-2 cytokines such as IL-4 or IL-10. The present study used two new synthetic prostaglandin E(1) (PGE(1)) analogs with oral bioavailability, misoprostol (MP), and enisoprost (EP), to determine if these agents (1) exert suppressive effects in vitro on cytokine production by fresh unseparated mouse splenocytes and (2) are beneficial in vivo when used in conditions mediated by excessive Th-1 or Th-2 cytokine production. Preliminary in vitro studies demonstrated that both MP and EP can inhibit mitogen-stimulated Th-1 and Th-2 cytokine production in a dose-dependent fashion. Interestingly, at low doses, a stimulatory effect on interferon-gamma production was seen for both agents. In vivo studies tested the ability of parenteral administration of MP to alter outcome in the parent-into-F1 model of acute or chronic graft-vs-host disease (GVHD), entities thought to be mediated by excessive Th-1 or Th-2 cytokine production, respectively. Administration of MP to mice undergoing acute GVHD resulted in little detectable effect. However, in three independent experiments, MP administration in chronic GVHD mice consistently blocked GVHD-associated lymphoproliferation. In two of three experiments, GVHD-associated autoantibody production was significantly reduced. Variability between individual mice and between experiments suggests that dosing regimens and MP preparation are of critical importance. Nevertheless, these findings raise the possibility that MP may be of benefit in the treatment of human diseases characterized by excessive Th-2 cytokine production and humoral autoimmunity, for example, human lupus.

Kinetics of Th1 and Th2 cytokine production during the early course of acute and chronic murine graft-versus-host disease. Regulatory role of donor CD8+ T cells.

Acute and chronic graft-versus-host disease (GVHD) in the parent-into-F1 model are mediated by predominantly cellular or humoral immune responses, respectively, and are strikingly different entities by 2 wk of disease. Both forms of GVHD, however, evolve from a common starting point, i.e., donor CD4+ T cell recognition of host alloantigen and IL-2 production. Our study examines the first 2 wk of GVHD to delineate the events that critically influence GVHD development. Surprisingly, both forms of GVHD are initially characterized by increased Th2 cytokine (IL-4 and IL-10) production and B cell activation which persists into wk 2. The earliest distinguishing features of acute GVHD were detectable at days 5 through 7 of disease and consisted of 1) expansion of donor CD8+ T cells, and 2) increased IFN-gamma production by donor CD4+ and CD8+ T cells. Interestingly, IFN-gamma production by donor CD4+ T cells was not seen if donor CD8+ T cells were not engrafted in comparable numbers. Chronic GVHD in the DBA-into-BDF1 model was found to be caused by a relative defect in the ability of DBA CD8+ T cells to induce acute GVHD and to produce IFN-gamma. These studies demonstrate that both acute and chronic GVHD begin as a Th2 cytokine-mediated, B cell stimulatory response. The transition to acute GVHD is critically dependent on the engraftment of donor CD8+ T cells, which terminate B cell hyperactivity by 1) eliminating activated B cells and 2) promoting IFN-gamma secretion by donor CD4+ T cells.

Decreased lymphocyte responses in free-ranging bottlenose dolphins (Tursiops truncatus) are associated with increased concentrations of PCBs and DDT in peripheral blood.

Since 1987, large-scale mortalities of dolphins have been reported along the Atlantic coast of North America, in the Gulf of Mexico, and in the Mediterranean Sea. Autopsied bottlenose dolphins, Tursiops truncatus, which were collected from the large-scale mortality along the Atlantic coast in 1987 to 1988, exhibited opportunistic infections indicative of immune dysfunction. Further, these animals had high levels of chlorinated hydrocarbons, such as PCBs and DDT, that can suppress immune functions. The purpose of this study was to determine whether there is a relationship between chemical contaminant exposure and immune response in free-ranging dolphins. In June of 1991, peripheral blood was obtained from members of a bottlenose dolphin population that resides along the west coast of Florida. Peripheral blood lymphocyte responses to Concanavalin A (Con A) and phytohemagglutinin (PHA) were determined in vitro and compared by regression analysis with contaminant concentrations in whole blood from a small subset of these animals (n = 5). These data indicate that a reduced immune response in these bottlenose dolphins was correlated with increasing whole blood concentrations of several contaminants. Specifically, inverse correlations were found between Con A-induced lymphocyte proliferation and tetrachlorinated to octachlorinated biphenyls (r2 values ranged from 0.70 to 0.87). Con A-induced lymphocyte responses also correlated inversely with p,p'DDT (r2 values of 0.73 and 0.79); o.p'-DDE (r2 values of 0.93 and 0.96); and p,p'-DDE (r2 values of 0.73 and 0.81).

1,2-Dibromoethane as an initiating agent for cell transformation.

The two-stage transformation assay increases the sensitivity of cells to chemicals and permits detection of carcinogens acting as initiating agents. 1,2-Dibromoethane, a representative halogenated aliphatic, has been tested in the two-stage BALB/c 3T3 cells transformation test at dosage from 16 microM to 128 microM. This dose range is much lower than those previously found efficient in transforming BALB/c 3T3 cells. Apart from the lowest dose, which induced borderline effects, all the other assayed dosages appeared to induce heritable changes in the target cells. The initiated cells were revealed as fully transformed foci both in the combination with a chronic promoting treatment and also by allowing cells to perform more rounds of cell replication. The results clearly show that 1,2-dibromoethane can act as an initiator of cell transformation.