Inflammatory arthropathy in MRL hematopoietic chimeras undergoing Fas mediated graft-versus-host syndrome.

OBJECTIVE: To determine whether overexpression of the Fas ligand (FasL) on activated lpr T lymphocytes could induce arthritic lesions when grafted into syngeneic wild-type MRL mice expressing normal Fas receptor levels. METHODS: Lethally irradiated MRL+/+ mice were reconstituted with congenic MRL/lpr bone marrow cells and splenocytes overexpressing FasL. Fas-mediated cytotoxic properties of repopulating lpr splenic lymphocytes were evaluated in vitro. Simultaneously, the hind paw ankles of the hematopoietic chimeras were histologically examined. RESULTS: The lpr lymphocytes repopulating the spleen overexpressed FasL and had in vitro Fas-mediated cytotoxic activity. Simultaneously, in vivo, articular (synovitis, pannus) and periarticular (periostitis) inflammation with bone resorption were observed. CONCLUSION: Arthritic lesions may be induced in Fas-expressing recipients by persistent engrafted syngeneic lymphocytes overexpressing FasL.

Fas-mediated liver damage in MRL hemopoietic chimeras undergoing lpr-mediated graft-versus-host disease.

Fas is an apoptosis-signaling receptor that triggers cell death upon binding to its ligand (FasL). Autoimmune-prone MRL/lpr mice, characterized by a spontaneous mutation of Fas, exhibit a defect in the activation-induced cell death of mature T cells through a Fas-mediated pathway. As a consequence of this defect, activated T cells accumulating in this strain overexpress the FasL and can therefore mediate in vitro Fas-dependent cytotoxicity. To determine whether hepatic injury could be the result of an interaction between T lymphocytes bearing FasL and Fas-expressing liver cells, the livers of lethally irradiated MRL+/+ recipients reconstituted with MRL/lpr lymphoid cells were studied. After transfer of MRL/lpr spleen cells, livers were infiltrated by polyclonal CD8+ T lymphocytes of lpr origin with a peak on day 21 postgrafting. These donor-derived intrahepatic lymphocytes overexpressed the FasL and exerted in vitro Fas-mediated cytotoxicity against Fas+ thymocytes, which was specifically inhibited by soluble recombinant Fas in a dose-dependent manner. These intrahepatic lymphocytes induced apoptosis in vitro, irrespective of MHC restriction, in Fas-expressing primary cultured hepatocytes. Histologic examination of the liver revealed severe endothelialitis as well as periportal and intralobular infiltrations of activated lymphocytes with apoptotic hepatocytes in their vicinity. Simultaneously, liver damage was ascertained by elevated serum transaminase levels. These observations support the notion that an Ag-independent mechanism involving FasL may play a role in certain liver pathologies.

MRL/lpr CD4- CD8- and CD8+ T cells, respectively, mediate Fas-dependent and perforin cytotoxic pathways.

Autoimmune-prone MRL/lpr mice, homozygous for the lpr mutation, exhibit defective apoptosis and develop generalized lymphoproliferation with the accumulation of a double-negative (DN: CD4- CD8-) T cell population. The capacity of lpr T lymphocytes to effectuate Fas- and perforin-mediated cytotoxicity was investigated. Spleen and lymph nodes cells spontaneously lyse Fas- targets (thymocytes) through a Fas-mediated mechanism as a consequence of their overexpression of Fas ligand (FasL) confirmed by semiquantitative reverse transcription (RT)-PCR and immunoprecipitation analysis. This cytotoxicity was greatly increased after stimulation of the effectors by phorbol myristate acetate (PMA) + ionomycin. Under these conditions, MRL/lpr spleen and LN cells exhibited strong Fas-mediated Ca2+-independent cytotoxic activity against wild-type Fas+ (H-2 compatible or incompatible) thymocytes or lipopolysaccharide (LPS)-transformed blast cells. Such Fas-mediated cytotoxic activity was also observed with C57BL/6-lpr, but never with wild-type C57BL/6 or MLR+/+ effectors. Depletion experiments showed that the effector cells of this Fas-mediated cytotoxicity were DN T cells. This subset, which represent in vivo activated T cells, can spontaneously lyse Fas+ targets by a mechanism that does not need the interaction of the T cell receptor (TCR) with major histocompatibility complex molecule plus antigen. This lytic potential is increased by PMA + ionomycin, which sends a second activation signal to these primed T cells. Therefore, the small amounts of Fas receptor expressed on MRL/lpr tissues may account for their nonspecific autoimmune attack by DN cells. In Con A-containing medium, which allows detection of the perforin-mediated pathway against Fas targets, cytotoxic CD8+ effectors were detected that are able to kill lpr thymocytes via a Ca2+-dependent pathway. Thus, in MRL/lpr mice, these CD8+ cells could constitute potent cytotoxic effectors against cells presenting antigen to their TCR.

Allogenic MHC class II determinant(s) in MRL/Ipr autoimmune disease-prone mice. Unusual expression of an L1 transposable element creates molecular mimicry.

In some aged MRL/Ipr autoimmune disease-prone mice, unexpected reactivity has been observed between lymphoid cells and mAb or polyclonal antibodies directed against the A beta d class II chain of the MHC. However, Southern blot analysis of their genomic DNA, using different class I and class II MHC-specific probes, confirmed their inbred character and their H-2k genotype. In this study, immunoprecipitation experiments with an anti-A beta d mAb indicated that the 45 (and 12)-kD molecule(s) recognized by an anti-A beta d mAb differed from a class II chain. After specific antibody screening of a lambda gt11 expression library constructed with MRL/Ipr "A beta d-positive" lymphoid cells, we cloned cDNA that share sequences with high homology (> 80%) to the 3' end of a 7-kb L1Md (L1) element propagated in the mouse genome via retrotransposition. Northern blot analysis showed an overtranscription of these L1 sequences in the MRL/Ipr spleen cells used for the construction of the cDNA library, in comparison to the MRL ancestor strains. Therefore, the autoimmune MRL/Ipr strain could express a translation product of L1 open reading frame 2, which mimics a "highly antigenic" epitope of an allogenic MHC class II Ag.

Imbalance of MHC class I expression in 3LL tumour cells.

Cell lines and a clone established from the C57BL/6 (H-2b) Lewis lung (3LL) tumour were previously characterized with respect to tumour growth and metastatic spread in vivo, and to the expression of a 3LL tumour-specific antigen (3LL TA) using a monoclonal antibody raised in syngeneic mice immunized with 3LL cells. No correlation was observed between the presence of 3LL TA and the prevention of metastatic spread which suggests that the immune recognition of this tumour antigen requires the presence of a self H-2 molecule absent from these tumour cells. Indeed, radioimmunoassay (RIA) and cytofluorometric analysis using specific monoclonal antibodies have shown that the H-2Kb molecule was not expressed at the cell surface of all 3LL cell lines and clones, while the H-2Db molecule was present at normal levels. This defect, which was not the consequence of a lack of beta 2m expression, was accompanied by an absence or a marked reduction of the H-2K mRNA level (which has been reversed in the M4 cell line by in vitro gamma interferon treatment), while the H-2D class I gene was normally transcribed. Another defective transcription was also observed for a gene in the Tla region (gene 37). This low '37' phenotype was corrected by in vitro treatment of the M4 cell line with gamma interferon, which indicates that this class I gene of the Qa/Tla region has an interferon response sequence in the promoter.

Immunogenetic studies of spontaneous abortion in mice. III. Non-H-2 antigens and gestation.

CBA/J (H-2k) females, mated with DBA/2 J (H-2d) or DBA/1 J (H-2q) males, exhibit a high rate of fetal resorption. In contrast, when H-2 identical CBA substrains (i.e. CBA/Ca and CBA/N) are used, this phenomenon is not observed. On the other hand, before mating with DBA/2 J males, pre-immunization of CBA/J females with spleen cells coming from BALB/c J or (DBA/2 x BALB/c J) F1 males (and not from other H-2d identical males whatever their Mls alleles) has significantly decreased the fetal resorption rate. Thus, immunization against determinants other than classical H-2d (K, I, D, L) antigens (transmitted as a dominant character and different from Mls determinants) can elicit anti-abortive effects. Furthermore, it was observed that the spleen cell endowed with the anti-abortive effects was neither a T nor a B lymphocyte. In contrast, peritoneal cells were able to reproduce the phenomenon, indicating that it may be mediated by a cell of the macrophage-monocyte lineage. Finally, a first gestation was substituted for allo-immunization of CBA/J females. The anti-abortive effects of a first pregnancy by BALB/c J males (and not by other H-2k syngeneic or H-2d allogeneic males) was observed in the course of a second pregnancy sired by DBA/2 J males. These data can be interpreted in terms of maternal recognition of an antigen present on both macrophages and trophoblast cells and necessary for a successful gestation, which is coded for by genes outside the K, I, D, L regions.

Further serological and RFLP analysis of the MRL-+/+ and MRL-lpr/lpr mice.

During their ageing, MRL-lpr/lpr mice (H-2k) suffer from progressive lymph node enlargement, associated with the development of several acute autoimmune lesions. The primary effect and location of the lpr mutation is unknown. However, a minority of the lymphoid cells of some MRL-+/+, as well as MRL-lpr/lpr mice, express 'alien' H-2d antigenic specificities. In the course of the investigation of the origin of the latter, we have carried out the genotyping of the MHC of several MRL-+/+ and MRL-lpr/lpr mice using the Southern blotting technique and employing a variety of MHC class I and class II probes, and restriction enzymes known to discriminate between the d and the k haplotypes. None of the results obtained so far suggests the contribution of genuine H-2d genes to the MHC of the MRL-+/+ and MRL-lpr/lpr mouse strains. Alternative hypotheses for the generation of the 'alien' epitopes are discussed.

In vivo and in vitro immune responses of CBA/N (Xid) mice against allogeneic cells.

CBA/N (Xid) mice classically present a genetically determined immune deficiency. This is characterized by the absence of certain B lymphocyte subpopulations (Lyb 3, 5, 7) and a hyporeactivity towards class II T independent antigens. These mice were examined in comparison with conventional CBA/Ca (H-2k) histocompatible controls for their immune reactivity, using different systems linked to allogenic transplantation: the local graft-versus-host reaction (LGVHR), allogeneic tumor (Sa 1 A/J) rejection, mixed lymphocyte reaction (MLR) and cell mediated lymphocytotoxicity (CML). It is concluded that a difference in reactivity is also present in some responses to classically T dependent antigens. This difference can result either in an increased reaction in the case of a primary LGVHR or a decrease in the same but late secondary reaction. The rejection of a tumor allograft (Sa 1 A/J - H-2a) is delayed in the CBA/N (H-2k) substrain with respect to conventional CBA mice. Cell mediated lymphocytotoxicity (CML) does not seem to be affected by the Xid defect in immunized cell donors. On the contrary, MLR responses are strikingly increased. It is suggested that the poor seeding of B cells into peripheral lymphoid organs alters the T/B ratio, favoring either T effector cells or T suppressors (Ts) according to the models used. Antibody responses to allogeneic H-2 antigens are not significantly modified.

Local active suppression and successful vaccination against spontaneous abortion in CBA/J mice.

We report here that vaccination of CBA/J female mice with DBA/2 X BALB/c recombinant line that decreases the spontaneous abortion rate increases local active decidua-associated suppressor cell activity. In contrast, vaccination with a recombinant line that increases the abortion rate decreases suppressor cell activity. No correlation was seen between the effect on the abortion rate and the ability of cells from the fetoplacental unit to inhibit cytolysis by NK cells. Successful vaccination against spontaneous abortion may act primarily by augmenting suppressor cell activity in the decidua at the implantation site.

Spontaneous autocytotoxicity against an unexpected H-2d haplotype in MRL/lpr (H-2k) autoimmune disease-prone mice.

The MRL/lpr (H-2k) inbred strain, a model for the autoimmune disease systemic lupus erythematosus, differs from the healthy inbred strain MRL +/+ (H-2k) by only 0.1% of its genome. Southern blot analysis using class I and class II probes confirmed the H-2k genotype of both strains. Among the Iak-positive peritoneal cells, cells with an unexpected expression of Iad specificities were detected in a radioimmunoassay using several monoclonal antibodies and one conventional antiserum. This was only found in aged (6- to 9-month-old) mice both in the MRL/lpr strain (32% Iad-positive mice) and in the MRL +/+ strain (42% Iad-positive mice). Furthermore, 24% of aged MRL/lpr mice exhibited strong spontaneous cytotoxic T lymphocyte (CTL) activities against P815 (H-2d) target cells, and 57% had a weaker but still detectable level of cytotoxicity. In contrast, such a CTL activity has never been found in the MRL +/+ strain. These results suggest that the anti-H-2d CTL plays a role in the onset of the autoimmune process in MRL/lpr mice.

Immunogenetic studies of spontaneous abortion in mice. II. Antiabortive effects are independent of systemic regulatory mechanisms.

CBA/J females (H-2k) mated with DBA2/J males (H-2d) exhibit a high rate of fetal resorption. Fetal survival has been improved by pretreatment of CBA/J females with spleen cells from BALB/c J (H-2d) (but not from CBA/J or DBA/2/J) males. Similarly, three out of nine recombinant inbred strains (recombining BALB/c and DBA2 genomes at the homozygous state) possess antiabortive effects like the BALB/c parent. Previous studies showed that BALB/c pretreatment triggers the appearance of suppressor cells in the spleen and of IgG1 anti-H-2d antibodies in the serum of CBA/J females. Studies of these two immunological parameters after CBA/J preimmunization by the different recombinant strains suggest that local intrauterine immunoregulation is the determinant of success or failure of allopregnancy.

Immunogenetic studies of spontaneous abortion in mice. Preimmunization of females with allogeneic cells.

CBA females (H-2k) mated with DBA2 males (H-2d) exhibit a high rate of fetal resorption (30%) when compared with the CBA female BALB/c male, CBA female/CBA male, DBA2 female/CBA male, DBA2 female/DBA2 male combinations (6 to 8%). Preimmunization of CBA females with spleen cells from DBA2, BALB/c, or CBA males were performed in order to test their effects on CBA maternal tolerance of (CBA X DBA2)F1 fetuses. Only preimmunization with BALB/c male cells was effective in decreasing resorption; cells from BALB/c females had no effect. In order to further test 1) the role of non-MHC-encoded antigens present in the BALB/c male background, 2) the necessity of an additional H-2 difference, and 3) whether or not the phenomenon is H-2d restricted, preimmunizations were performed by using cells from congenic BALB/k (H-2k), BALB/b (H-2b), or BALB/c (H-2d). Only the latter treatment was efficient, which suggests that the paternal H-2d haplotype must be presented in synergy with some non-MHC-encoded antigens in the BALB/c male background. Immunogenetic studies with cells from nine recombinant inbred strains that reassorted DBA2 and BALB/c genomes showed that three of them behave like BALB/c and six like DBA2. This would suggest that the genetic determinism of this phenomenon is simple.

Immunologic consequences of vaccination against abortion in mice.

CBA/J female mice have a high rate of fetal resorption when mated with DBA/2J males. This fetal wastage can be dramatically reduced by immunizing the female with BALB/cJ but not DBA/2J spleen cells. We report here that immunization with BALB/cJ (but not DBA/2J) spleen cells leads to 1) anti-paternal MHC antibody that is predominantly of the IgG1 isotype, and which disappears from the serum during pregnancy; 2) increased active suppression in both the spleen and placenta; and 3) an ability to adoptively transfer the fetal protection and placental suppression with serum from the immunized mice. Congenic absorption studies before adoptive transfer indicate that the active component of the serum is also directed against the paternal MHC haplotype. These results indicate that maternal humoral immunity can lead to increased fetal protection in correlation with local active suppression in the placenta. They also suggest an expansion of the placental immunoabsorbent hypothesis to include the induction of active suppression against maternal cell-mediated immunity.

Heterogeneity in surface antigen of cell lines and clones from a murine carcinoma.

Cell lines of the murine Lewis lung tumor (3LL) were established from primary tumor site (PT) and from lung metastatic foci (Met). Expression of histocompatibility antigens was assessed by immunofluorescence with an anti-H-2b antibody and by tumorigenicity of the cells in different strains of mice. Analysis for tumor-associated transplantation antigens (TATA) were performed by challenging mice with living tumor cells after pretreatment with irradiated cells or by in vivo tumor neutralization assays with different tumor cells as target cells and T lymphocytes from immunized animals as effector cells. The 3LL tumor was found to be heterogeneous in respect to tumor growth rate in vivo, metastatic capacity in vivo, expression of normal histocompatibility antigens and expression of "TATA". Moreover, monoclonal antibody B1 (H11-115) binding to a Met cell surface antigen, but not to normal cells, was obtained. The antibody bound to 3LL tumor cell lines from primary tumor (PTH, PTV), from a mixture of metastatic foci (MetH, MetV), from different metastatic foci originating from 3 different mice, and to 21 subclones derived from one of the lines established from a metastatic focus. Analysis of the 3LL sublines demonstrated a significant antigenic heterogeneity within each tumor population as well as among the different sublines.

Vaccination against spontaneous abortion in mice.

We report here that the high rate of spontaneous resorption observed in CBA/J female mice mated with DBA/2 J males can be dramatically reduced by vaccination with Balb/c male spleen cells, but not by CBA/J or DBA/2 J male spleen cells. This effect correlates with the differential ability of Balb/c spleen cells to induce MLR suppressor activity in CBA/J female mice, and should lead to a better understanding of the immunology of the materno-fetal relationship.

Immunoprecipitation of insulin receptors by antibodies against Class 1 antigens of the murine H-2 major histocompatibility complex.

Insulin receptors from C57BL/6J mouse (H-2b) liver membranes were specifically labeled with 125I-photo-reactive insulin by UV irradiation. Membranes were solubilized and the capacity of various antibodies reacting with the major histocompatibility complex to immunoprecipitate insulin receptors was tested. About 5% of the labeled receptors were immunoprecipitated by a conventional mouse antiserum against H-2b histocompatibility antigens and by a monoclonal antibody against Class 1 antigens of the H-2b haplotype (Kb and Db). No immunoprecipitation was obtained with a monoclonal antibody against Class 2 antigens of I-Ab or against Class 1 antigens of the H-2k haplotype. Insulin receptors can thus be specifically immunoprecipitated by antibodies against class I histocompatibility antigens.

Prevention of graft-versus-host mortality in mice by preincubation of the graft with highly purified spleen-derived immunosuppressive peptides.

Two non-species-specific spleen-derived immunosuppressive peptides (SDIP) have been highly purified from bovine spleen and are referred to as SDIPe and SDIPHP with reference to the last step of the purification procedure (electrophoresis or high pressure liquid chromatography, respectively). Preincubation of lymphoid cells with very dilute preparations of both SDIPs significantly reduced their capacity to induce a lethal graft-versus-host reaction (GVHR), but did not diminish their capacity to form hematopoietic colonies in the spleens of lethally irradiated syngeneic hosts (CFUs). Therefore, such pretreatment of bone marrow grafts by SDIP could be useful candidate for GVHR prevention. In contrast, preincubation with thymulin (serum thymic factor (FTS) plus ZnCl2), which shares several physicochemical properties with SDIP, significantly improved the ability of lymphoid cells to induce lethal GVHR. The opposite immunomodulatory effects of these highly similar peptides are discussed.

Comparative analysis of the immunopharmacological properties of three new nitrosourea analogues: RPCNU, RFCNU and Chlorozotocin.

Three sugar derivatives of chloroethyl nitrosoureas: RPCNU, RFCNU and Chlorozotocin (CLZ) were examined for their effect on the immune system in mice when administered at the minimal antineoplastic therapeutic dose. When the drugs were administered 4 days prior to antigenic stimulation, a potentiation of DTH to oxazolone was observed whereas the antibody response to SRBC was markedly decreased. When injected 24 h after immunization, the nitrosourea analogues did not modify the intensity of DTH reaction and of allograft rejection and only RFCNU depressed the antibody response. The proliferative response of spleen cells to T or B cell mitogens was strongly depressed after in vivo treatment with either of the three analogues. Concomitantly, peritoneal macrophages became strongly cytostatic for tumor cells and also displayed an enhanced chemiluminescence upon phagocytosis. An augmentation of antibody-dependent cellular cytotoxicity of spleen cells was observed only after CLZ administration which also resulted in an increase of NK activity of peritoneal cells but in an inhibition of spleen cell cytotoxicity. This study demonstrates that the nitrosourea analogues act as immunomodulating agents and reinforces their interest as antineoplastic drugs since they can stimulate nonspecific effector mechanisms known to play a role in resistance against tumors.

In vitro and in vivo specificities of purified thymic factors inhibiting graft-versus-host reaction.

A calf thymic factor has been prepared by extraction in distilled water, followed by selective precipitation with ethanol and ammonium sulfate. The non-dialysable fraction obtained by this method was further purified by a preparative electrofocusing technique which allows the isolation of two fractions which can specifically inhibit, in vitro, without toxicity or species specificity, spontaneous DNA synthesis in hemopoietic cell suspensions. When injected in vivo in mice, 3 days after immunization with SRBC, both these fractions exhibited immunosuppressive properties. In contrast, they were without any effect on a thymus-independent antibody response to TNP-LPS, whatever the day of administration. Still more important, in vitro pretreatment of lymphoid cells by the purified fractions inhibits their ability to elicit a graft-versus-host reaction when injected to histo-incompatible recipients.

Sensitivity of cloned high- and low-metastatic murine Lewis lung tumor cells to lysis by cytotoxic autoreactive cells.

Cloned malignant cell lines from primary tumor sites (two lines) and from a lung metastatic focus (one line) were established 14 and 28 days after s.c. inoculation of Lewis lung tumor cells into C57BL/6 mice. Cloning was done in semisolid agarose culture medium and individual clones expanded in liquid medium. In vivo malignancy of the three lines was assured by graft experiments to healthy C57BL/6 recipients. Morphology, cloning efficiency in agarose, and growth rate were the same for all three lines. However, the cloned line from a metastatic focus had a higher metastatic rate (number of lung metastases per 10(5) injected tumor cells) compared to the two other cell lines. T-lymphocyte-depleted mononuclear spleen cells from mice grafted with tumor cells 2 to 4 weeks previously were found to be cytotoxic to cells from all three lines, whereas unfractionated mononuclear spleen cells from the same animals had weak or no cytotoxic activity. The cytotoxicity of T-lymphocyte-depleted spleen cells was found to include other malignant and nonmalignant target cells of C57BL/6 origin, but not allogeneic cells. In mixing experiments, splenic T-lymphocytes inhibited the cytotoxic activity of non-T-lymphocytes. The high- and low-metastatic tumor cell lines were found to be equally sensitive to lysis by T-cell-depleted spleen cells, suggesting that the effector cells (cytotoxic autoreactive cells) may have a significant antineoplastic potential.