Antibodies to early pregnancy factor retard embryonic development in mice in vivo

Previous work in this laboratory has shown that passive immunization of mice against early pregnancy factor (EPF) leads to failure to maintain pregnancy. The findings presented in this paper demonstrate that this treatment affects the development of the embryos very early in gestation. By Day 3, 54 and 25% of embryos in the 2 groups treated with anti-EPF immunoglobulin (Ig)G and IgM, respectively, had not developed to the 4-cell stage, compared with 12 and 1% in the control groups. None of the embryos in the mice treated with anti-EPF had developed beyond the 8-cell stage. A similar delay in development after treatment was observed on Day 4. The effect apparent during the early stages of cleavage is an indirect rather than a direct one, as 2-cell embryos (32-36 h post coitum), cultured in vitro in the presence of anti-EPF antibodies, developed uninterrupted to the morula and blastocyst stage. The delay in development did not appear to be caused by a disruption of the normal pattern of circulating progesterone, as progesterone concentrations on Day 4 were within the normal range for Quackenbush mice.     

Immunotherapy of murine sarcomas with auto-anti-idiotypic monoclonal antibodies which bind to tumor-specific T cells

Hybridomas producing monoclonal antibodies (mAb) were obtained from BALB/c mice immunized against either of two transplanted, chemically induced syngeneic sarcomas, MCA-1490 or MCA-1511. Two mAb, 4.72 and 5.96, were obtained, one from each immunization. They were found to have apparent anti-idiotypic specificity in that they, when injected s.c., primed naive BALB/c mice for delayed-type hypersensitivity that was specific for the immunizing tumor and required homology at genes linked to the Igh-1 allotype locus. Neither mAb bound tumor antigen. When mice with established transplants of MCA-1490 or MCA-1511 were treated by repeated i.p. injections of the appropriate anti-idiotypic mAb (4.72 and 5.96, respectively), a significant reduction in tumor growth was observed in those mice that had received the appropriate mAb. The idiotope defined by mAb 4.72 was expressed by T cells in mice responding to MCA-1490. mAb 4.72 bound to T cell suppressor factors that were specific for MCA-1490 and were derived from T cell hybridomas or sera of mice bearing MCA-1490. mAb 4.72 also bound to cells from lymph nodes draining the area of a growing MCA-1490 tumor. It was used, in combination with cell sorting, to establish a T cell line, which mediated delayed-type hypersensitivity to MCA-1490 and inhibited the outgrowth of MCA-1490 in BALB/c mice. Thus, mAb specific for idiotopes on T cells responding to syngeneic tumor antigen had both direct immunotherapeutic activity and could be used to establish cultures of tumor-reactive T cells.     

Relationship between the effectiveness of the antitumor action of sarcolysine and the latency period between transplantation and tumor appearance

Sarcoma induced with 3-methylcholantrene in mice was transplanted to syngeneic recipients. The latent period of the tumour growth varied from 7 to 26 days. Sarcolysine was injected once when the tumour became palpable. When the tumours were revealed 7 to 8 and 10 to 15 days after the inoculation, the tumour growth inhibition was accompanied by increased survival of mice, particularly marked at the latter period. When the tumours were revealed 17 to 20 or 22 to 26 days after the transplantation, inhibition of the tumour growth was not accompanied by any increase of survival.     

Relationship between early pregnancy factor, mouse embryo-conditioned medium and platelet-activating factor.

The effects of synthetic platelet-activating factor (PAF-acether) and mouse embryo-conditioned medium (a source of embryo-derived PAF (EPAF)) on production of early pregnancy factor (EPF) were compared. Embryo-conditioned medium, itself inactive in the EPF bioassay, stimulated ovarian production of EPF in vitro but PAF-acether did not. In vivo, embryo-conditioned medium induced EPF activity in serum of oestrous female, but not in male, mice in contrast to PAF-acether, which induced activity in serum of both male and female mice. This PAF-induced activity was transitory, declining significantly by 2 h and disappearing by 3 h after injection. Activity induced by embryo-conditioned medium was first evident at 2 h after injection, serum concentrations increasing up to 6 h after injection. By discriminating between the behaviour of PAF-acether and EPAF, these studies reinforce the conclusions of other workers that the molecule produced by the embryo is not PAF. Further investigations into the mechanism of action of PAF-acether revealed that it is a potent inducer of activity in the EPF bioassay, with an absolute requirement for platelets in the spleen cell suspension used in the assay. This platelet-derived active species was bound specifically by an anti-EPF monoclonal antibody, indicating that it is EPF-like. This is consistent with parallel studies showing that platelets are not required for induction of activity by either pregnancy serum or purified EPF. These studies were applied to the PAF-induced leukotriene-like species, which had been found by others to be active in the EPF bioassay. Pregnancy serum induced the appearance of this substance from the spleen cell suspension used in the assay; thus the leukotriene-like substance may be regarded as an effector molecule in vitro or mediator of the initiating stimulus of EPF in the bioassay.     

Activation of a functional idiotype network response by monoclonal antibody specific for a virus (M-MuLV)-induced tumor antigen

BALB/c mice were injected with IgM mAb specific for Moloney murine leukemia virus (M-MuLV)-determined cell surface Ag in an attempt to inhibit Moloney sarcoma growth. The monoclonal IgM significantly inhibited sarcoma growth when given to the mice after inoculation with Moloney murine sarcoma/leukemia virus, and also potentiated the in vivo antibody response specific for M-MuLV Ag. These responses were significantly greater than the primary response to the virus alone in age- and sex-matched control mice, and were also seen in mice which were injected with the IgM antibody only and not with virus, suggesting that an Ag-independent mechanism may be involved. The M-MuLV-specific serum antibody responses induced by the monoclonal IgM, with or without prior virus inoculation, were predominantly of the IgG1 isotype, with some IgG2a; no other isotypes were found to have titers significantly higher than in the normal response to virus alone. M-MuLV-specific IgG1 was detected only in mice injected with monoclonal IgM, and not in the response to virus alone. The same sera also had high titers of anti-idiotypic antibodies, (Ab2), as well as anti-anti-idiotypic antibodies (Ab3). It appears, therefore, that passive immunization with M-MuLV-specific IgM mAb activates an idiotypic network, which results in both Ab2 and Ab3 responses; the M-MuLV-specific response may be considered a subset of Ab3.     

IgM but not IgG monoclonal anti-Nocardia brasiliensis antibodies confer protection against experimental actinomycetoma in BALB/c mice

Nocardia brasiliensis is a facultative intracellular microorganism that produces a human chronic infection known as actinomycetoma. Human and mouse anti- N. brasiliensis antibody response identify P24, P26 and P61 immunodominant antigens. In this work, we generated immunoglobulin M (IgM) and IgG monoclonal antibodies (mAbs) specific to immunodominant P61 antigen. The monoclonal IgM (NbM1) and IgG2a (NbG1) antibodies were assessed for their in vitro bactericidal activity, in vivo protective effect and ability to block catalase activity. These mAbs specifically recognized P61, but they did not inhibit its enzyme activity. The in vitro bactericidal effect of NbG1 was higher than the killing ability of the IgM mAb. In vivo experiments with a murine model of experimental infection with N. brasiliensis injected into rear footpads was used to test the effect of NbM1 and NbG1. The negative untreated group developed a chronic actinomycetoma within 4 weeks. IgM mAbs conferred protection to BALB/c mice infected with N. brasiliensis . IgG mAb lacked this protective effect. IgM mAb showed a dose–response correlation between antibody concentration and lesion size. These results demonstrate that humoral immune response mediated by antigen-specific IgM antibody protects against an intracellular bacterial infection.     

Characterization of the antigen identified by Po66

Summary The mouse monoclonal antibody (mAb) Po66 has been shown in previous work to be localized in nude mice xenografts of human lung tumours when injected intravenously [Dazord L et al. (1987) Cancer Immunol Immunother 24: 263–268] and to be suitable for the scintigraphic detection of lung cancers in patients [Dazord L, et al. (1987) in Klapdor (ed) New tumour markers and their monoclonal antibodies. Georg Thieme, Stuttgart, New York, pp 444–450]. The nature of the antigen recognized by Po66 has been investigated in the present work and comparisons are made with antigens recognized by other mAbs prepared in the laboratory. These mAbs were raised either against lung squamous cell carcinoma (mAbs Po43, Po60), or against a bronchio-alveolar carcinoma (mAbs BAM33, BAM45, BAM54 and BAM69). Radioiodinated purified Po66 did not compete for cell binding with any other mAb. All Po and BAM mAbs reacted with tumour cells both cultured in vitro and grown in vivo. They recognized cytoplasmic antigens as judged by immunofluorescence examination of fixed cells or by immunoperoxidase staining of cancer tissues, but could never be visualized by immunofluorescence on the surface membrane of culture cells. The mAbs of the BAM series reacted with vimentin as demonstrated by immunofluorescence staining, showing alterations in the aspect of the filaments under the effect of colchicin. Radiolabelled mAbs Po43, BAM33 and BAM45 bound to partially purified cytoplasmic cytoskeleton components. In contrast, Po66 was never seen associated with intermediary filaments. The sensitivity to enzyme digestion of the antigen associated with Po66 was studied in comparison with those associated with Po43, BAM33 and BAM45. All antigens were sensitive to protease digestion while only the Po66-identified antigen was sensitive to periodate, neuraminidase and -fucosidase. Thus, mAb Po66 identified an antigen of 47 kDa (as determined before) present in the cytoplasm but not related to the cytoskeleton, not detected on the cell surface and glycoproteic in nature.     

Immunotherapy of B lymphoma by anti-idiotype antibodies: Characterization of variant tumour cells appearing a long time after the initial tumour inoculation

Summary Immunotherapy of a murine B-cell lymphoma by antibodies to idiotypic determinants of its IgM, resulted in surviving tumour-free mice. Several of these mice, however, did develop tumours a long time after the initial inoculation of the tumour cells, or upon rechallange of the survivors with B-lymphoma cells. The presence of tumour cells during this long latent period may have been due to the development in the host of an anti-idiotype immune response. These late-developing tumours were detected by a radioimmunoassay and characterized by immunofluorescent staining and sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Cells of some late-developing tumours lost the idiotype recognized by the antibodies used for the immunotherapy. Several of these tumours expressed IgM on the cell surface, while others did not, because of the absence of light chains. They were identical in the structure of their rearranged µ chain genes proving their derivation from the original inoculation. Cell lines obtained from the late-developing tumours differed in their tumorigenicity. The appearance of idiotype-negative tumour cells as a result of anti-idiotype immunotherapy has a great impact in our efforts to cure lymphoma by this modality.     

In vitro and in vivo effects of an anti-mouse endoglin (CD105)–immunotoxin on the early stages of mouse B16MEL4A5 melanoma tumours

TGF-beta superfamily co-receptors are emerging as targets for cancer therapy, acting both directly on cells and indirectly on the tumour neovasculature. Endoglin (CD105), an accessory component of the TGF-beta receptor complex, is expressed in certain melanoma cell lines and the endothelial cells of tumour neovessels. Targeting endoglin with immunotoxins is an attractive approach for actively suppressing the blood supply to tumours. Here, we report evidence indicating that endoglin is expressed in mouse melanoma B16MEL4A5 and mouse fibroblast L929 cell lines. We prepared an immunotoxin to target endoglin by coupling the rat anti-mouse MJ7/18 (IgG2a) monoclonal antibody (mAb) to the non-toxic type 2 ribosome-inactivating protein nigrin b (Ngb) with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) as a linker with a molar nigrin b at a MJ7/18 stoichiometry of 2:1. The MJ7-Ngb immunotoxin generated killed both cell lines, with IC₅₀ values of 4.2 × 10^?9 M for B16MEL4A5 and 7.7 × 10^?11 M for L929 cells. For in vivo assays of the immunotoxin, B16MEL4A5 cells were injected subcutaneously into the right flanks of 6-week-old C57BL/6 J mice. When the animals developed palpable solid tumours, they were subjected to treatment with the immunotoxin. While treatment with either MJ7/18 mAb or Ngb did not affect tumour development, treatment with the immunotoxin completely and steadily blocked tumour growth up to 7 days, after which some tumours re-grew. Thus, vascular-targeting therapy with this anti-vascular immunotoxin could promote the destruction of newly created tumour vessels at early stages of B16MEL4A5 tumour development and readily accessible CD105+ B16MEL4A5 melanoma cells.     

Hepatitis induced by an IgM monoclonal antibody against procarboxypeptidase R

Procarboxypeptidase R (proCPR), also known as thrombin-activatable fibrinolysis inhibitor (TAFI), is present in plasma and can be activated to carboxypeptidase R (CPR) by trypsin-like enzymes such as thrombin and plasmin. CPR has the carboxypeptidase B-like activity that can inactivate the inflammatory peptides such as C5a by removing the C-terminal arginine and can interfere with fibrinolysis by removing C-terminal lysine residue of fibrin. In the present study, we conducted to produce monoclonal antibodies (mAbs) by using spleen cells from proCPR-deficient mice immunized by partially purified mouse proCPR. The mAbs obtained were IgM isotype and reacted with proCPR and interfered with activation of proCPR to CPR by thrombin-thrombomodulin complex. Some BALB/c mice implanted with the hybridoma died in 7 days, and intravenous injection of the mAb to BALB/c mice induced transient elevation of GOT and GPT in plasma although injection to the deficient mice did not. Furthermore, the histological features showed the focally lesions in liver tissue of BALB/c mice injected with the mAb. Since liver is the major site of proCPR synthesis, IgM mAb to proCPR should have induced local inflammation at the side resulting in induction of hepatitis.     

Differences between the catabolism and tumour distribution of intact monoclonal antibody (791T/36) and its Fab/c fragment in mice with tumour xenografts revealed by the use of a residualizing radiolabel (dilactitol-125I-tyramine) and autoradiography

Summary Radioiodine-labelled 791T/36 monoclonal antibody (mAb) and its Fab/c fragment, consisting of one Fab arm and the Fc portion, have identical whole-body survival curves in BALB/c mice (t1/2 = 3.75 days). Therefore, these two forms of this antibody provide a suitable model for studying the role of valency in the targeting efficiency of antibodies to tumours in vivo. 791/T36 antibody and its Fab/c fragment were labelled either by direct iodination using the iodogen method (¹²⁵I) or by dilactitol-¹²⁵I-tyramine (¹²⁵I-DLT), a residualizing label, which accumulates in the cells involved in degradation of the carrier protein. In tumour-bearing nude mice, the percentage of injected dose of mAb or Fab/c fragment reaching the specific 791T tumour was similar, and these proteins appeared to be catabolized at a similar rate in this tissue. mAb, but not the Fab/c fragment, was found to be very actively catabolized by the liver and spleen of tumour-bearing mice compared to control nude mice, this probably resulting from clearance of immune complexes. This effect was most pronounced when the mAb was labelled with¹²⁵I-DLT, the percentage of injected dose of mAb reaching the spleen and liver being higher than the percentage of injected dose reaching the tumour. This effect was not seen with the Fab/c fragment. Autoradiographic studies on tumour sections, which exhibit antigenic sites throughout the tumour mass, showed that the Fab/c fragment was already homogeneously distributed in the tumour 12 h after injection whereas the whole antibody was mainly localized at the periphery of the tumour. Those results suggest a binding site barrier effect. Overall, these results indicate that the highest valency and affinity may not be the optimal choice for mAb to be used for therapeutic purposes.     

The in vitro and in vivo effects of anti-galactose antibodies on endothelial cell activation and xenograft rejection

We have previously produced a series of antigalactose (anti-Gal) hybridomas and characterized their heavy chain gene usage. Here we have quantified the affinity of these Abs for the alpha-Gal epitope and characterized their in vitro effects on endothelial cell activation and apoptosis. We report that anti-Gal mAbs derived from Gal(-/-) mice show a range of affinity for the alpha-Gal epitope, and that affinity was generally increased as the V(H) gene usage transitioned from germline sequences to sequences exhibiting somatic maturation. Despite an 85-fold range in affinity, all the anti-Gal mAbs examined induced alpha-Gal-specific endothelial cell activation, and after prolonged exposure induced endothelial cell apoptosis in a complement-independent manner. Only murine anti-Gal mAbs of the IgM or IgG3 subclass, but not IgG1, were effective at initiating complement-dependent cell lysis. Using a novel rat to mouse xenograft model, we examined the in vivo ability of these mAbs to induce xenograft rejection and characterized the rejection using histology and immunohistochemistry. Infusion of complement-fixing IgG3 mAbs resulted in either hyperacute rejection or acute vascular rejection of the xenograft. Surprisingly, infusion of an equal amount of a high affinity anti-Gal IgG1 mAb, that fixed complement poorly also induced a rapid xenograft rejection, which we have labeled very acute rejection. These studies emphasize the importance of in vivo assays, in addition to in vitro assays, in understanding the role of anti-Gal IgG-mediated tissue injury and xenograft rejection.     

Evaluation of human carcinoembryonic-antigen (CEA)-transduced and non-transduced murine tumors as potential targets for anti-CEA therapies

The MC-38 C57BL/6 mouse colon adenocarcinoma cell line has been transduced with a retroviral construct containing cDNA encoding the human carcinoembryonic antigen (CEA) gene [Robbins PF, Kantor JA, Salgaller M, Horan Hand P, Fernsten PD, Schlom J (1991) Cancer Res 51: 3657]. Two clones, MC-38-ceal and MC-38-cea2, expressed high levels of CEA on their cell surface. A third CEA-expressing cell line, MCA-102-cea3, was similarly derived by transduction of the MCA-102 C57BL/6 mouse fibrosarcoma cell line and is described here. In this study, the three CEA-transduced murine tumor cell lines (MC-38-cea1, MC-38-cea2, MCA-102-cea3) were evaluated for their tumorigenic potential, as well as their ability to serve as in vivo model systems for active and passive immunotherapy studies. Parameters that were investigated include tumor growth rate, the antibody response of the host to CEA, and the CEA content of the tumors. The MC-38-cea2 model appeared to be the most appropriate for immunotherapy studies. Biodistribution studies, using an¹²⁵I-labeled anti-CEA mAb, demonstrated efficient tumor targeting of MC-38-cea2 tumors in C57BL/6 and athymic mice.     

[F-18]-Fluoro-2-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-glucose positron emission tomography as a tool for early detection of immunotherapy response in a murine B cell lymphoma model

[F-18]-fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET) is a non-invasive imaging technique which has recently been validated for the assessment of therapy response in patients with aggressive non-Hodgkin’s lymphoma. Our objective was to determine its value for the evaluation of immunotherapy efficacy in immunocompetent Balb/c mice injected with the A20 syngeneic B lymphoma cell line. The high level of in vitro FDG uptake by A20 cells validated the model for further imaging studies. When injected intravenously, the tumour developed as nodular lesions mostly in liver and spleen, thus mimicking the natural course of an aggressive human lymphoma. FDG-PET provided three-dimensionnal images of tumour extension including non-palpable lesions, in good correlation with ex vivo macroscopic examination. When mice were pre-immunized with an A20 cell lysate in adjuvant before tumour challenge, their significantly longer survival, compared to control mice, were associated with a lower incidence of lymphoma visualized by PET at different time points. Estimation of tumour growth and metabolism using the calculated tumour volumes and maximum standardized uptake values, respectively, also demonstrated delayed lymphoma development and lower activity in the vaccinated mice. Thus, FDG-PET is a sensitive tool relevant for early detection and follow-up of internal tumours, allowing discrimination between treated and non-treated small animal cohorts without invasive intervention. C. Chaise and E. Itti contributed equally to the work.     

Regulation of Ia+ reticulum cell sarcoma (RCS) growth in syngeneic SJL/J mice. I. Inhibition of tumor growth by passive administration of L3T4 monoclonal antibody before or after tumor inoculation

Spontaneously arising reticulum cell sarcoma (RCS) tumors in SJL/J mice stimulate syngeneic host T lymphocytes to proliferate and are dependent on host T cells for maintenance and growth. Tumor-associated Ia antigens have been implicated in the proliferative response both in vivo and in vitro, and the responding T cells are predominantly Lyt-1+2- L3T4+. We hypothesized that elimination or depletion of the responding L3T4 subpopulation in vivo should inhibit growth of transplantable RCS tumors, and continued RCS growth may be dependent on the continued presence of L3T4 cells. This hypothesis was tested experimentally by examining the effect of passive administration of L3T4 monoclonal antibody (mAb) into SJL/J mice either before or at different times after tumor inoculation. The tumor inoculum used killed all mice 15 to 30 days after injection. Administration of a single dose of L3T4 mAb 4 days before tumor inoculation resulted in complete depletion of L3T4 cells and complete inhibition of tumor growth. The antibody-treated mice survived with no sign of tumor growth even after complete recovery of L3T4+ cells. These results demonstrate that initiation of tumor growth is dependent on host L3T4+ cells. Administration of mAb as late as 7 days after tumor inoculation resulted in inhibition of tumor growth, and administration of mAb at day 10 resulted in significant inhibition of tumor growth. Compared with the kinetics of tumor growth in normal control mice, administration of L3T4 after tumor inoculation results in tumor growth arrest. These findings demonstrate that continued tumor growth in vivo is dependent on the presence of L3T4+ cells. In the RCS system, the present studies show that administration of mAb to L3T4+ cells is therapeutic in that it inhibits the induction of tumor growth, and it also prevents tumor growth in tumor-bearing animals.     

Pathogenic natural antibodies propagate cerebral injury following ischemic stroke in mice

Self-reactive natural Abs initiate injury following ischemia and reperfusion of certain tissues, but their role in ischemic stroke is unknown. We investigated neoepitope expression in the postischemic brain and the role of natural Abs in recognizing these epitopes and mediating complement-dependent injury. A novel IgM mAb recognizing a subset of phospholipids (C2) and a previously characterized anti-annexin IV mAb (B4) were used to reconstitute and characterize injury in Ab-deficient Rag1(-/-) mice after 60 min of middle cerebral artery occlusion and reperfusion. Reconstitution with C2 or B4 mAb in otherwise protected Rag1(-/-) mice restored injury to that seen in wild-type (wt) mice, as demonstrated by infarct volume, demyelination, and neurologic scoring. IgM deposition was demonstrated in both wt mice and reconstituted Rag1(-/-) mice, and IgM colocalized with the complement activation fragment C3d following B4 mAb reconstitution. Further, recombinant annexin IV significantly reduced infarct volumes in wt mice and in Rag1(-/-) mice administered normal mouse serum, demonstrating that a single Ab reactivity is sufficient to develop cerebral ischemia reperfusion injury in the context of an entire natural Ab repertoire. Finally, C2 and B4 mAbs bound to hypoxic, but not normoxic, human endothelial cells in vitro. Thus, the binding of pathogenic natural IgM to postischemic neoepitopes initiates complement-dependent injury following murine cerebral ischemia and reperfusion, and, based also on previous data investigating IgM reactivity in human serum, there appears to be a similar recognition system in both mouse and man.     

Tumour rejection after adoptive transfer of line-10-immune spleen cells is mediated by two T cell subpopulations

Summary The growth of line-10 tumours in naive guinea pigs is prevented by adoptive transfer of spleen cells that are hyperimmune to this hepatocellular carcinoma. To study the T cell subpopulations responsible for the adoptive transfer of immunity, various cell populations were removed from immune spleen cells using monoclonal antibodies (mAbs) and magnetic microspheres. Spleen cell subpopulations were identified by mAb after flow cytometry and rosette formation with the magnetic microspheres. mAb CT5 was confirmed to be a pan T cell marker, while the CT6 (anti-T-suppressor/cytotoxic) and CT7 (anti-T-cell) markers were present on two different T cell subpopulations. So our results show that CT7 mAb cannot be used as a pan T cell marker as was published previously. Moreover, the mAb H155 (anti-T-helper/inducer) reacted with the same T cell subpopulation recognized by CT7. So we designated this H155/CT7-positive subpopulation as T helper/inducer cells. Removal of the CT6-, CT7-, or the H155-positive T cells from the immune spleen cells resulted in loss of the in vitro poliferative response to line-10 tumour protein and tuberculin purified protein derivative (PPD). The H155/CT7(anti-T-helper/inducer)-positive spleen cells did not express MHC class II antigens as determined by mAb 25E3. In most experiments, elimination of MHC-class-II-positive cells did not change the in vitro proliferative response to line-10 protein, whereas the response to tuberculin PPD was completely abrogated. Immune spleen cells after depletion of CT6-, CT7- or H155-positive cells, failed to transfer immunity. However, after depletion of MHC-class-II-antigen-positive cells the line-10 immunity was still present, whereas the immune response to tuberculin PPD was lost. In conclusion, our data indicate that immunity to the line-10 tumour is the result of a cooperation between at least two different T cell subpopulations, the T helper/inducer (CT7/H155) cells and the T suppressor/cytotoxic cells (CT6). If this is a common feature, then the therapeutic approach of in vitro expanded TIL cells should take into consideration the requirement of two T cell subsets.     

Immunoglobulin M efficacy against Cryptococcus neoformans: mechanism, dose dependence, and prozone-like effects in passive protection experiments

The IgM mAbs 12A1 and 13F1 are protective and nonprotective, respectively, against lethal Cryptococcus neoformans infection in mice. To better understand the variables that contribute to IgM efficacy against C. neoformans, we studied the effects of inoculum size, route of infection, and Ab dose for each of these mAbs. mAb 13F1 did not prolong survival under any condition studied. mAb 12A1 prolonged survival after the administration of certain Ab doses after i.p. infection with defined inocula and promoted phagocytosis, agglutination, and the formation of inflammatory cell rings around yeast cells in vivo. Large Ab doses of mAb 12A1 resulted in either no protection or enhanced infection, consistent with a prozone-like effect. Investigation of this phenomenon revealed that the fungal cell was protected against microbicidal nitrogen-derived oxidants when large amounts of Ab were bound to the C. neoformans capsule. mAb 12A1 was opsonic in vitro for peritoneal, but not splenic or alveolar macrophages. In summary, our results indicate that IgM efficacy against C. neoformans is a function of the route of infection, inoculum, and Ab dose and is associated with its ability to promote opsonization, agglutination, and phagocytic ring formation in vivo. The occurrence of the prozone-like phenomenon implies that high Ab titers are not necessarily beneficial in assuring protection against certain pathogens and that caution should be exercised in using high Ab titer as a measure for vaccine efficacy.     

In vivo regulation of the murine syngeneic mixed lymphocyte reaction

Previous work from this laboratory has suggested that a CD8+ T suppressor (Ts) cell network regulated the murine syngeneic mixed lymphocyte reaction (SMLR). We have attempted to disrupt this network by the inoculation of anti-CD8 monoclonal antibodies (mAb) in vivo. Intraperitoneal inoculation of three mAbs resulted in a marked increase in the proliferation of CD4+, self-Ia-reactive splenic T cells in vitro to syngeneic, but not to allogeneic, spleen cells. Suppression was not limited to a specific mouse strain as the enhanced SMLR was reproducible following anti-CD8 treatment of three strains of mice. In vivo depletion of CD8+ T cells was not a prerequisite for enhancement of the SMLR as several mAb to CD8 augmented the SMLR independent of their capacity to cause CD8 T cell depletion. Moreover, enhancement of the SMLR could be mimicked in vitro by inclusion of anti-CD8 mAb in in vitro cultures of responder T cells and irradiated Ia+ syngeneic stimulators. Since the in vitro SMLR was enhanced following mAb treatment, it was expected that the in vivo SMLR would also be increased. However, no evidence of increased in vivo autoreactivity could be detected following in vivo treatment with anti-CD8 mAb, indicating that other mechanisms in addition to CD8+ regulatory T cells acted to regulate the in vivo activity of autoreactive T cells.