Impairment of germline transmission after blastocyst injection with murine embryonic stem cells cultured with mouse hepatitis virus and mouse minute virus.

The aim of this study was to determine the susceptibility of murine embryonic stem (mESCs) to mouse hepatitis virus (MHV-A59) and mouse minute virus (MMVp) and the effect of these viruses on germline transmission (GLT) and the serological status of recipients and pups. When recipients received 10 blastocysts, each injected with 10(0) TCID(50) MHV-A59, three out of five recipients and four out of 14 pups from three litters became seropositive. When blastocysts were injected with 10(-5) TCID(50) MMVp, all four recipients and 14 pups from four litters remained seronegative. The mESCs replicated MHV-A59 but not MMVp, MHV-A59 being cytolytic for mESCs. Exposure of mESCs to the viruses over four to five passages but not for 6 h affected GLT. Recipients were seropositive for MHV-A59 but not for MMVp when mESCs were cultured with the virus over four or five passages. The data show that GLT is affected by virus-contaminated mESCs.

Canine stem cell factor augments expression of matrix metalloproteinase-9 by CD34 cells.

BACKGROUND: Canine models have proved to be predictive of clinical findings in human bone marrow (BM) transplantation; consequently, the utilization of dogs is an excellent tool for supporting therapeutic purposes. Considering the role of growth factors in homing and mobilization of hematopoietic progenitors, the aim of this work was to evaluate whether canine stem cell factor (cSCF) contributes to matrix metalloproteinase (MMP)-9 secretion by CD34 cells. METHODS: The study was carried out in a cell population selected by immunomagnetic techniques using the anti-canine CD34 monoclonal antibody (MAb) 3B4 produced by us. Secretion of MMP-9 was evaluated by zymography. RESULTS: Analyzes of canine CD34(+) cells guaranteed that the MAb 3B4 was optimum for selecting a subset population with defined characteristics of primitive hematopoietic cells. The isolated cells were able to proliferate onto irradiated pre-established stroma, giving rise to mature neutrophils. There was also a 20-fold enrichment in the long-term culture-initiating cell content when the isolated population was added to irradiated cultures, with respect to the starting mononuclear cell population. DISCUSSION: We have provided the first evidence that canine BM CD34(+) cells constitutively express MMP-9 and the role of cSCF in up-regulating the secretion of this enzyme. The fact that cSCF augments expression of MMP-9 together with the ability of the isolated CD34(+)cells to proliferate onto irradiated pre-established stroma enables further investigations to determine whether the secretion of MMP-9 mediated by cSCF is one of the factors that enhance migration, homing and repopulation of primitive hemopoietic cells.

The Fc-region of a new class of intact bispecific antibody mediates activation of accessory cells and NK cells and induces direct phagocytosis of tumour cells.

Bispecific antibodies (bsAb) are considered as promising tools for the elimination of disseminated tumour cells in a minimal residual disease situation. The bsAb-mediated recruitment of an immune effector cell in close vicinity of a tumour cell is thought to induce an antitumoural immune response. However, classical bispecific molecules activate only a single class of immune effector cell that may not yield optimal immune responses. We therefore constructed an intact bispecific antibody, BiUII (anti-CD3 x anti-EpCAM), that not only recognizes tumour cells and T lymphocytes with its two binding arms, but also binds and activates Fcgamma-receptor positive accessory cells through its Fc-region. We have demonstrated recently that activated accessory cells contribute to the bsAb-induced antitumoural activity. We now analyse this stimulation in more detail and demonstrate here the BiUII-induced upregulation of activation markers like CD83 and CD95 on accessory cells and the induction of neopterin and biopterin synthesis. Experiments with pure cell subpopulations revealed binding of BiUII to CD64+ accessory cells and CD16+ NK cells, but not to CD32+ B lymphocytes. We provide further evidence for the importance of the Fc-region in that this bispecific molecule stimulates Fcgamma-R-positive accessory cells to eliminate tumour cells in vitro by direct phagocytosis.

Analysis of peripheral immune tolerance uncovers a mouse strain-dependent in situ type of graft tolerance.

We screened various mouse strains [C57BL/6, BALB/c, DBA/2, CBA/Ca, (CBAxC57L/6)F1, SJL, C3H] for induction of peripheral immune tolerance. Only CBA/Ca mice treated with anti-CD4 + CD8 monoclonal antibodies and grafted with allogeneic skin showed long-term graft survival (150 to >200 days). Interestingly, T cells from the tolerant CBA/Ca mice rejected bone marrow/spleen cells of the skin graft donor strain and caused lethal graft-versus-host disease when transplanted to the donor strain. Furthermore, peripheral tolerance was easily broken: CBA/Ca mice could be reactivated to reject their tolerated grafts via immunization with (graft donor x recipient strain)F1 bone marrow cells. Thus, in contrast to the generalized nature of central tolerance, our experiments show that peripheral immune tolerance is strain dependent and locally restricted to graft tissue.

Long-lasting unresponsiveness to polyclonal T cell-binding immunoglobulins.

We have shown that mice after a single injection of anti-T cell antibody followed by multiple injections of a second xeno-, allo- or syngeneic anti-T cell antibody differing from the former in species origin developed specific, long-lasting tolerance to the second antibody. To characterize the mechanism of this anti-antibody unresponsiveness and the modalities accompanying the preinjection step, we injected mice with anti-pan T, anti-CD4 or anti-CD8 monoclonal antibodies, followed by multiple injections of polyclonal rabbit anti-mouse thymocyte globulin (RbATG). Our observations indicate that: (i) Depletion of CD4+ cells is the most important factor for tolerance induction to subsequently injected RbATG. Non-depleting mAb were less effective, and antibody-induced CD4 modulation or blockade were inconsequential. (ii) The prevention of anti-antibody responses involves specific B cell tolerance, as shown by suppression of anti-RbATG but not anti-bovine serum albumin (BSA) antibodies after challenge of tolerant mice with RbATG-BSA conjugates. (iii) Suppression of anti-antibody responses involves T cell unresponsiveness rather marginally, as demonstrated by in vitro spleen cell restimulation with RbATG and in vivo antibody response to RbATG-fluorescein isothiocyanate hapten conjugate. (iv) Analysis of isotypes of anti-RbATG antibodies does not suggest alterations in Th1/Th2 tuning as being responsible for this kind of tolerance. (v) Over 150-day survival of fully allogeneic skin grafts (CBA-to-C57BL/6) was observed in mice preinjected with anti pan-T or anti-CD4 mAb followed by RbATG. Mice treated with anti-CD4 mAb alone rejected allografts within 21 days and induced anti-antibodies. Taken together, our experiments suggest B cell tolerance as main mechanism in this type of acquired long-term humoral unresponsiveness to foreign, polyclonal, T cell-binding immunoglobulins.

Induction and suppression of anti-antibodies to syngeneic T cell-binding antibodies in mice.

Considerable effort is being invested in antibody gene technologies for production of species-adapted anti-T cell antibodies which can overcome formation of neutralizing anti-antibodies (anti-Ab). By establishing a mouse model for the generation of syngeneic anti-T cell MoAb, we addressed the question of whether ideally species-adapted T cell-binding antibodies can prime mice to produce anti-Ab. Two anti-Thy-1.2 MoAbs of IgG2a (MmTC) or IgM (MmTC-IgM) isotype were generated in congenic C57B1/6-Thy-1.1 mice. Three injections of MmTC in C57B1/6-Thy-1.2 or (C57B1/6-Thy-1.2xC57B1/6-Thy-1.1)F1 hybrids where they act as syngeneic anti-T cell MoAbs induced low anti-Ab. When MmTC was injected as Igh-mismatched antibody in CBA/J mice, high titre anti-MmTC antibodies were measured and fully mismatched skin allografts rejected within 26 days. MmTC injected twice weekly in syngeneic C57B1/6 mice induced low anti-MmTC and prolonged graft survival up to day 117. In contrast, retreatment induced anti-MmTC with graft rejection within 32 days. Thus, upon retreatment, the immunosuppressive effects of MmTC were inhibited to a similar extent as that seen after antibody treatment in Igh-mismatched mice. However, a recently analysed immunological principle to suppress anti-Ab against T cell binding allo- or xenoantibodies by preinjection of T cell-depleting antibody with species differences in heavy chains also suppressed syngeneic anti-Ab after MmTC retreatment. This effect was accompanied by prolonged graft survival. Thus, our data indicate that inhibitory anti-Ab must be considered in humans even when treated with fully species-adapted anti-T cell MoAb, but may be suppressed by preinjection of a Fc region-mismatched anti-T cell antibody.

Preclinical evaluation of biotin labeling for red cell survival testing.

Biotin labeling of red cells was tested in dogs as a preclinical study for cell survival. Red cells were labeled with either spacered Biotin-X-NHS (BxNHS) or water-soluble biotin compounds. After reinfusion, biotinylated red cells were detected in small blood samples (5 microliters) with flow cytometry. Improved BxNHS labeling allows an easy detection of positive red cells for almost 100 days, whereas labeling with watersoluble compounds-despite strong labeling during the first days-results in a decrease of label, which prevented a discrimation between labeled and negative cells after about 4 weeks. When biotin labeling of red cells was compared with 51Cr labeling, slopes of red cell survival were quite similar after the latter were corrected for elution. Survival slopes were linear, and the mean survival time was t = 93 d. In two blood-donor dogs the slopes of red cell survival where log linear and the mean survival time was t = 45 d. In conclusion, BxNHS, but not the water-soluble biotin compounds, is a good nonradioactive, nontoxic alternative for red cell survival studies. No health hazards are to be expected from the very low dose of Dimethylformamide, which is used as a solvent for biotin-x-NHS.

Comparison of T-cell subpopulations in cats naturally infected with feline leukaemia virus or feline immunodeficiency virus.

T-cell subsets were studied by flow cytometry in 58 feline leukaemia virus (FeLV)-positive cats with naturally acquired FeLV infection to determine whether the changes in CD4+ or CD8+ T cell populations differed from those observed in 55 feline immunodeficiency virus (FIV)-positive cats with naturally acquired FIV infection. The sole criterion for inclusion into the study was seropositivity. Mean (SD) CD4+ T cell values of FeLV positive cats were decreased to 31.1 (8.0) per cent and their CD8+ T cell values were increased to 22.8 (6.3) per cent in comparison with uninfected control cats (37.9 [9.5] per cent CD4+; 15.2 [6.3] per cent CD8+). The CD4+/CD8+ ratio was reduced to 1.5 (0.7), compared with 3.0 (1.5) in 39 FeLV-and FIV-negative control cats. Differences from control values were significant, but there was no significant difference between CD4+ and CD8+ lymphocytes of FeLV-versus FIV-infected cats. These findings indicate that FeLV and FIV have similar effects on T lymphocyte subsets. Both retrovirus infections can induce immunodeficiency, both viruses infect a broad range of lymphohaemopoietic cells, despite having different primary target cells, and can induce the killing of lymphocytic cells in vitro. It is concluded that a decreased CD4+/CD8+ ratio is not restricted to FIV infections but may also occur in FeLV infection.

Immunosuppression by Fc region-mismatched anti-T cell antibody treatment.

Formation of anti-antibodies (anti-Ab) is known to counteract immunotherapy with anti-T cell antibodies. Our previously described immunological approach prevented anti-Ab with the consequence of prolonged survival of fully mismatched skin grafts in C57BL/6 mice. These mice were treated with a single priming injection of a monoclonal anti-T cell Ab followed by repeated injections of anti-T cell mAb differing in species origin from the priming mAb. We now show prolonged tolerance to discordant xenogeneic, to bispecific, and even to polyclonal Ab, and demonstrate that the underlying immunosuppressive principle is due to a difference in heavy chain constant region between first and second antibodies, independent of whether or not they share the same idiotype. To examine this phenomenon, a panel of mAb was generated which share the same mouse anti-Thy-1.2 idiotype, but carry a human IgG1(T23), IgG3(T212C8), or mouse IgG2a(MmT1) constant heavy chain region. We found that sequential injection of MmT1 and T23 according to the above treatment schedule induced huIgG1 isotype-specific tolerance to T23, which was similar to that seen when using a primary mAb (MmT5) that was, instead, fully mismatched with T23 in both idiotype and constant region. Thus, differences of idiotype between primary and booster Ab were inconsequential for their ability to inhibit anti-Ab formation. This novel form of induced specific tolerance to anti-T cell Ig survived graft rejection and was still evident 230 days after termination of the T cell depletion protocol. Taken together, these results demonstrate that rechallenge with Fc region-mismatched Ab opens an immunological window that allows for induction of tolerance to immunogenic anti-T cell Ab and prolonged immunosuppression.

Immunological approach to inhibit formation of anti-antibodies to allo- and xenogeneic anti-T cell immunoglobulin.

Inhibitory anti-antibodies induced in patients by xenogeneic or even by humanized anti-T cell antibodies remain an unresolved problem. Mice also produce anti-antibodies following injection of xeno- or allogeneic anti-T cell antibodies. Here we report a principle based on sequentially applied anti-T cell antibodies generated in different species, which results in suppressed anti-antibody formation and prolonged immunosuppression. Thus, a single priming injection in mice of mouse (MmT1 or MmT5 differing by idiotype only) or of rat (RmT1) anti-mouse Thy-1 monoclonal antibodies (mAb) or of rat anti-mouse L3T4 + Ly-2 (RmCD4 + CD8) mAb suppressed anti-antibody formation against subsequent booster injections of one of the above antibodies, provided that they differed in species origin from the priming antibody. Correspondingly, a sixfold and longer prolongation of 50% survival of fully mismatched skin grafts was observed. Less or no anti-antibody suppression and little prolongation of graft survival was obtained if the 'first' and the 'second' (and following) antibody injections were of the same species, differing by iso- or idiotype only. Finally, the suppressive principle did not manifest itself at all if the initial antibody injection included both the first and second antibody. These findings are discussed with reference to earlier studies on hapten/carrier effects as well as on immunosuppression attributed to 'non-depleting' rat anti-CD4/CD8 T cell antibodies.

Biotin labeling as an alternative nonradioactive approach to determination of red cell survival.

Biotin labeling of red cells was studied using different approaches to see if biotinylation is a useful label for determination of erythrocyte survival. Mouse red cells were labeled with biotin, either in vivo by injection or in vitro. In vivo labeled red cells were followed up in some mice without transfusing the labeled erythrocytes. Furthermore, in vivo labeled as well as in vitro labeled red cells were transfused into syngeneic mice. The biotin label allows an easy discrimination between labeled and unlabeled red cells during FACS analysis, and it is relatively stable for at least 50 days. All the three different approaches give similar results. Mean red cell life spans of in vivo or in vitro labeled red cells either transfused or followed up in vivo were between 44 and 52 days (T50 mean 23.9 days) when red cell destruction was assumed to be only a result of senescence. Mean red cell life spans were between 8 and 18 days (T50 mean 9.5 days) when a random destruction independent of red cell age was suggested. All the survival slopes are neither simple linear functions of time nor logarithmic functions, but they show an overlay of linear function by a logarithmic function where the components of both are unknown.

Antigen binding and effector functions of a chimeric antibody with a deletion of the CH1 domain and non-covalently associated kappa chains.

In a mouse/human chimeric IgG1 Ab with a deletion of the CH1 domain the disulfide bridges between H and L chain cannot be formed, although the corresponding cysteine residues are present. We show that the kappa chains are non-covalently associated to H chain dimers and that they can dissociate from the H chains. Therefore different populations of Ab can be distinguished according to the number of associated kappa chains. Only the molecules with two kappa chains can bind to the target cell. Since this Ab was derived from a T cell depleting anti-Thy-1.2 Ab, we can assess the implications of this conformational alteration as to binding avidity, effector functions and immunosuppression in vivo.

Murine anti-mouse T cell monoclonal antibodies elicit anti-antibodies in mice: intra-species immunization model for estimating potential patient sensitization against humanized anti-T cell antibodies.

Humanization of immunosuppressive anti-T cell monoclonal antibodies (mAb) raises the question as to how completely it helps to avoid formation of neutralizing anti-antibodies (anti-Ab) in patients. To get more information on intra-species sensitization against anti-T cell mAb, we produced two immunosuppressive mouse IgG2a anti-mouse Thy-1.2 mAb (MmT1 and MmT5) in AKR/J mice and measured the potential of MmT1 to elicit inhibitory anti-Ab in AKR/J (H-2k), C57BL/6 (H-2b), congenic B10.BR (H-2k) and DBA/2 (H-2d) mice. After one injection once weekly for 4 weeks of 5 micrograms MmT1 (200 micrograms/kg) in C57BL/6 mice, without the use of any adjuvants, high concentrations of anti-Ab directed against MmT1 (300 micrograms/ml) and MmT5 (100 micrograms/ml) were measured by enzyme-linked immunosorbent assay. Similar concentrations of anti-Ab were found in immunized DBA/2 and less in B10.BR mice. No syngeneic anti-Ab could be produced in AKR/J. From the C57BL/6 mice, we raised anti-MmT1+, MmT5- idiotype (IDIO1) and anti-MmT1+, MmT5+ allotype (ALLO1) mAb. An in vivo test system was adapted to measure the inhibitory effects of circulating poly- or monoclonal anti-Ab. It revealed a reduction of in vivo depletion capacity not only of the sensitizing mAb (MmT1), but also of another anti-Thy-1.2 mAb (MmT5), with identical allotype but different idiotype. From this we conclude that intra-species immunization following injection of anti-T cell mAb can produce high titer inhibitory anti-idiotype and anti-allotype antibodies. Implications for hyperchimeric or fully human anti-T cell mAb are discussed.

Antilymphocytic antibodies and marrow transplantation. XII. Suppression of graft-versus-host disease by T-cell-modulating and depleting antimouse CD3 antibody is most effective when preinjected in the marrow recipient.

A hamster antimouse CD3 monoclonal antibody (MoAb) opened the way to experimental studies on the suppression of allograft rejection and cytokine-related morbidity after treatment with antibodies modulating the CD3/T-cell receptor complex (CD3/TCR). Because earlier attempts to suppress graft-versus-host disease (GVHD) in patients by in vitro treatment of donor marrow with anti-CD3 MoAb had remained inconclusive, we used a rat IgG2b antimouse CD3 MoAb (17A2) with fewer side effects to analyze suppression of GVHD in the mouse model. Detailed phenotyping of blood, spleen, and lymphnode T cells after the injection of 400 micrograms 17A2 in C57BL/6 mice showed 60% CD3 downmodulation and 50% T-cell depletion for spleen cells. Injection of these spleen cells, together with bone marrow cells, in fully mismatched preirradiated CBA mice delayed GVHD by only 6 days. Ex vivo treatment of donor cells with 17A2 was not effective. In contrast, conditioning of marrow recipients with a single injection of 17A2 delayed 50% GVHD mortality by 100 days and prevented GVHD altogether after prolonged treatment, with survivors showing complete chimerism and specific transplantation tolerance. This difference in antibody effect contrasts with earlier experiences with nonmodulating but more strongly T-cell-depleting MoAbs of the same isotype, which prevent GVHD no matter whether applied in vitro or injected into donor or recipient mice. Our data indicate that CD3/TCR reexpression in marrow recipients with no circulating 17A2 is the reason why ex vivo donor cell treatment with anti-CD3 MoAb is comparatively ineffective. Our data, which allow separate evaluation of cell-depleting and cell-modulating antibody activity, help to explain previous clinical failure to suppress GVHD and provide evidence in favor of conditioning the marrow recipient with anti-CD3 MoAb as a therapeutic alternative.

Decline in CD4+ cell numbers in cats with naturally acquired feline immunodeficiency virus infection.

T-cell subsets were studied by fluorescence-activated cell sorter analysis in 57 feline immunodeficiency virus (FIV)-seropositive cats with naturally acquired FIV infection to see whether CD4(+)-CD8+ alterations were comparable to those observed in human immunodeficiency virus-infected patients. CD4+ values were decreased and CD8+ values were increased. The CD4+/CD8+ ratio was reduced to 1.6, compared with 3.3 in 33 FIV-seronegative control cats. Variance analysis of data showed a significant influence of FIV seropositivity, sex, and spaying of female cats on CD4+ values. CD8+ values were significantly influenced by FIV seropositivity, age, and breed. These findings indicate a similarity between FIV and human immunodeficiency virus infections, as far as alterations of T-cell subsets are concerned.

[T-helper and T-suppressor lymphocyte subpopulations in the peripheral blood of spontaneously FIV-positive cats]. / T-Helfer- und T-Suppressor-Lymphozyten-Subpopulationen im peripheren Blut spontan FIV-positiver Katzen.

Reduced CD4(+)-T-helper-lymphocyte- and increased CD8(+)-T-suppressor-lymphocyte-subsets were found in peripheral blood of 47 FIV-seropositive cats with naturally acquired FIV-infection. The CD4+/CD8+ ratio was decreased, too. Variance analysis of data included the variables reaction in FIV-test, age group, and race. Similarities with HIV-infection were discussed.

Functional characterization of canine lymphocyte subsets.

Functional characterization of subsets of T lymphocytes is essential for transplantation studies in dogs, as it is in other species. We studied the function of T cells separated by two mouse monoclonal antibodies recognizing complementary subsets--an antibody directed to canine T cells (MdT-P1) with an up-regulating function, and an antibody directed to human CD 8 (MT811) that cross-reacts with down-regulating canine T cells. Immunorosetting with sheep red blood cells and Percoll gradient allowed us to study depleted and enriched fractions. Their function was tested in mixed lymphocyte culture (MLC), cell-mediated cytotoxicity (CML), and coculture with B cells in a hemolytic plaque assay (PFC). In MLC, MdT-P1-positive cells showed a high proliferative response, and MT811-positive cells responded poorly to allogeneic cells. Vice versa, MT811- negative cells responded strongly, and MdT-P1-negative cells were poor responders but strong stimulators. Effector cells of CML were separated following 8 days of culture and prior to mixing with target cells. Enriched and depleted fractions with either antibody showed low cytotoxic activity as compared with unseparated cells. When added to unseparated effector cells MT 811-positive cells suppressed cytotoxicity. B cells were obtained by rosetting with staphylococcal protein A (SPA). Their immunoglobulin production was studied following 6 days of culture stimulated by pokeweed mitogen in a reverse hemolytic plaque assay. Again, MT 811-positive cells added to the culture suppressed, and MT 811-negative cells enhanced immunoglobulin production. In conclusion, immunorosetting with two monoclonal antibodies allowed us to distinguish subpopulations of canine T cells with up-regulating (helper/inducer) from those with down-regulating (suppressor) activity.