Search

Modification of tumor cells with one or more costimulatory adhesion molecules has been proposed as a means to develop therapeutic cancer vaccines for use in human immunotherapy. Expression of B7-1 (CD80) in tumors by gene transfer creates an immunogenic tumor cell that induces antitumor immunity and protects mice from further challenge with wild-type tumor cells. In this report, we demonstrate that protein transfer of glycosyl-phosphatidylinositol (GPI)-anchored costimulatory molecules into tumor cell membranes could be used as an alternative to gene transfer for tumor immunotherapy. Incubation of isolated tumor membranes with purified GPI-anchored B7-1 results in stable incorporation of B7-1 on tumor cell membranes within a few hours. Immunization of C57BL/6 mice with EG7 tumor membranes modified to express GPI-B7-1 by protein transfer induces tumor-specific T-cell proliferation and CTLs. In addition, immunization with these EG7 membranes protects mice from parental tumor challenge. The protein transfer approach used here does not require foreign vectors or live tumor cells and is completed within a matter of hours. Irradiated cells or membrane preparations from fresh or frozen tumor tissue can be used. Therefore, protein transfer of glycolipid-anchored molecules provides an efficient and novel approach to modify tumor membranes for human immunotherapy. This approach is not limited to costimulatory molecules because any cell surface protein can be converted to a GPI-anchored form by recombinant techniques.

The costimulatory molecule B7-1 (CD80) has been shown to be an important component for T cell immune responses. We have generated several monoclonal antibodies (PSRM-1, -2, -3, -6, and -7) against B7-1 using a human glycosylphosphatidylinositol-anchored B7-1 (GPI-B7-1) as an antigen. These monoclonal antibodies are able to detect B7-1 by flow cytometry, ELISA, and Western blotting. One antibody in particular, PSRM-3, blocks the CD28/CTLA-4 interaction with B7-1 and consequently blocks costimulation of T cells. The other PSRM monoclonal antibodies did not compete with PSRM-3 for recognition of B7-1 and also failed to block B7-1 interaction with CTLA-4 and CD28, indicating that these antibodies bind to different epitopes. PSRM-3 and -7 detect phosphatidylinositol-specific phospholipase C-released soluble GPI-B7-1 in a sandwich ELISA. We used this sandwich ELISA to assay for the presence of a soluble form of B7-1 in synovial fluids of arthritis patients. By sandwich ELISA, B7-1 was detected in the synovial fluid of 5/11 patients with rheumatoid arthritis, 5/5 patients with osteoarthritis, and 2/6 patients with other forms, including crystalline-induced arthritis. The presence of soluble B7-1 was confirmed by immunoprecipitation using PSRM-3-coupled Sepharose beads. The source and function of soluble B7-1 are unknown at present; it is possible, however, that the soluble form of B7-1 molecule may play a local immunoregulatory role which may suppress or induce inflammation depending upon whether it interacts with the T cell costimulatory CD28 molecule or the negative signaling CTLA-4 molecule.

The X-linked immunodeficiency (Xid) in CBA/N mice serves as a model for the X-linked agammaglobulinemia (XLA) syndrome in man. X-chromosome inactivation in F1 heterozygotes derived from CBA/N (Xxid/Xxid) and B6.Pgk-1a (X+/Y) was investigated by monitoring the methylation status of the individual Pgk-1 alleles, Pgk-1b and Pgk-1a, respectively, using a novel Tth111I RFLP. Results indicate that in circulating B lymphocytes of female heterozygotes, only the X chromosomes carrying the normal alleles (X+) are active (nonrandom inactivation of the X chromosome), whereas in non-B cells both the X chromosomes (X+ and Xxid) are active (random inactivation of the X chromosome). These results were further confirmed by direct evaluation of transcription of the Btk gene, the gene mutated both in Xid and in XLA.

Recently mouse models have shown that expression of costimulatory molecules such as B7-1 on tumor cells can induce tumor-specific immunity, suggesting that tumor cells modified to express costimulatory molecules can be a potential tumor vaccine. To investigate the importance of B7-1 co-stimulation in induction of autologous tumor immunity in humans, we established a renal carcinoma cell line, RCC-1, from a tumor resection and studied the patient's antitumor immune responses in vitro. The RCC-1 cell line constitutively expressed major histocompatibility complex (MHC) class I, intercellular adhesion molecule (ICAM)-1, and leukocyte function-associated antigen (LFA)-3 molecules, and MHC class II molecules were induced by interferon-gamma (IFN-gamma) treatment in vitro. However, neither RCC-1- nor IFN-gamma-treated RCC-1 cells expressed B7-1, and both failed to induce T-cell proliferative responses in mixed lymphocyte and tumor cell reaction (MLTR) assays, suggesting that the costimulatory signals provided by cell adhesion molecules such as ICAM-1 and LFA-3 were not sufficient to elicit an antitumor immune response. However, on transfection of the human B7-1 into RCC-1, these cells were able to induce a significant T-cell proliferation in MLTR assays. This T-cell response could be blocked by anti-B7 mAb treatment of the tumor cells. RCC-1B7 cells also induced the generation of tumor-specific cytolytic T lymphocytes to the parent RCC-1 cells in vitro, with little nonspecific cytolysis of an unrelated RCC line, A498, or autologous phytohemagglutinin (PHA) blasts. This specific cytotoxicity could be abrogated by anti-CD8 mAb and complement treatment. In summary, our study indicates that B7-1-CD28 interaction plays a critical role in induction of autologous tumor-specific cytotoxic T lymphocytes (CTLs) in humans, suggesting that the costimulatory molecule transfected tumor cells could be useful in expanding tumor-specific autologous CTL in vitro for adoptive tumor immunotherapy.

Background: Treatment with interleukin-2 (IL-2) and lymphokine-activated killer cells (LAK) resulted in responses in some patients with advanced renal cell carcinoma (RCC). However, the relative therapeutic benefit of the addition of LAK to IL-2 was unknown., Methods: A randomized Phase III trial was conducted in patients with RCC comparing continuous intravenous infusion (CI) IL-2 alone with CI IL-2 plus LAK. Interleukin-2 was administered at 3 x 10(6) U/m2/day on days 1-5, 13-17, 21-24, and 28-31. Patients on the LAK treatment arm underwent leukapheresis on days 8-10 and LAK cell reinfusion on days 13-15. The results are reported with long-term follow-up. The published experience with IL-2 alone or with the addition of LAK was investigated in a quantitative literature survey. The response proportions were studied by schedule (high dose bolus, moderate dose, low dose) and by concomitant administration of LAK., Results: Seventy-one patients were treated, 36 on the IL-2 arm and 35 on the IL-2 plus LAK arm. Four patients (6%) had major responses (two complete, two partial). The median survival of all patients was 13 months (95% confidence interval [CI], 9-18 months). There were no differences between treatment arms with regard to response (P = 0.61) and survival (P = 0.67). More patients on the LAK arm experienced pulmonary toxicity (P = 0.008). The overall weighted response proportion was 16% (95% CI, 8%-24%) for the 39 published series of 1291 patients treated with IL-2. The 95% confidence intervals for response proportion overlapped when compared by schedule and by administration of LAK., Conclusions: The dose and schedule of IL-2 used in this study resulted in a low level of antitumor activity and the addition of LAK did not improve the response rate against RCC. Given the infrequent, but reproducible, responses with IL-2 and interferon-based regimens, continued investigation of these agents is warranted as is the study of new cytokines. Alternative treatment strategies should be studied in RCC and new agents and treatment regimens that appear promising in Phase II studies must be studied in randomized trials.

Purified glycosyl phosphatidyl inositol (GPI)-anchored cell surface proteins can be reincorporated spontaneously into the cell membrane by incubating the cells with these proteins. This unique property provides a novel way of introducing cell surface receptors on live cell membranes without the use of gene transfection. Since any classical transmembrane cell surface protein can be converted to a GPI anchored protein by recombinant techniques, this method provides a means of studying ectodomain associated receptor functions on various cell types. Moreover, in some circumstances, it can be used to correct deficient cellular functions resulting from lack of cell surface protein expression. Using GPI-anchored Fc gamma receptor III (CD16B), a low affinity Fc gamma receptor, we have systematically studied the optimal conditions for reconstitution of a functional receptor on nucleated cells. CD16B is purified to homogeneity from neutrophil lysates by single step immunoaffinity chromatography. The purified CD16B is functionally active as evidenced by its ability to bind IgG opsonized erythrocytes. CD16B incorporation on nucleated cells is temperature dependent with an optimum of 37 degrees C. The level of expression of incorporated CD16B is also depend on the concentration of CD16B available and the duration of incubation. The incorporated CD16B retains its ability to bind ligand and also mediates endocytosis of the bound ligand. In summary, our results demonstrate that purified, functionally active GPI-anchored receptors can be expressed on desired cells in a controlled manner and retain some functional properties.

Background: Histological evaluation of serial endomyocardial biopsies performed at fixed time intervals after cardiac transplantation is the universal method used for the detection of cardiac rejection and assessment of the adequacy of antirejection therapy. No noninvasive methodology thus far investigated has achieved a high enough sensitivity and predictive accuracy to be considered as a potential replacement for endomyocardial biopsy in the detection of rejection in adults. The present study exploited the finding that the rate of spontaneous mutation in the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene is higher in proliferating human T cells than in resting cells. Thus, it was reasoned that in the posttransplantation setting, the frequency of HPRT- cells in peripheral blood may provide an indirect measure of alloactivated T lymphocytes., Methods and Results: This study consisted of determining the clonal frequency of HPRT- mutant cells (FMC/10(6) peripheral blood mononuclear cells [PBMCs]) within a total of 293 peripheral blood samples representing various numbers of sequential samples from each of 27 transplant recipients. These sequential samples represented time periods when endomyocardial biopsy specimens showed either (1) no evidence of rejection (n = 5 patients), (2) a single initial episode after transplantation of early (< 1 year) or late (> 1 year) rejection (n = 12 patients), or (3) multiple rejection episodes (n = 10 patients). Statistical analyses were used to quantify the time profiles of FMC/10(6) PBMCs in serial samples among transplant recipients and to determine the association of these profiles with both the onset of first rejection episodes and, in appropriate patients, the recurrence of rejection episodes. Data showed that PBMCs from patients with no evidence of rejection uniformly gave low values of < 6 FMC/10(6) cells, a frequency similar to that seen in healthy nontransplanted volunteers. In contrast, 19 of the 22 PBMC samples that were obtained from patients whose corresponding biopsy sample was diagnosed with a histological rejection grade of > or = 3 gave values of > 6 FMC/10(6) cells, 11 of which gave values > 50/10(6) cells (range, 146 to 46,982 FMC/10(6) cells). A significant association between the onset of first rejection and an increased rate of FMC/10(6) values was noted (P = .0001). The ability of a rising trend in FMC/10(6) values to correctly identify the onset of rejection was 81.8% and to correctly identify no rejection, 100%. In addition, a significant association between recurrent rejection episodes and persistence of high FMC/10(6) values in the weeks after treated rejection episodes was noted (P = .0003). The ability of a persistently elevated trend in values of FMC/10(6) cells to correctly identify recurrent rejection was 90% and to correctly identify no rejection, 100%., Conclusions: Increasing frequencies of HPRT- mutant cells in peripheral blood correlated with the onset of first rejection, and persistently elevated HPRT- mutant cells in the weeks after a treated rejection episode correlated with recurrent rejection. This quantitative noninvasive assay may thus serve as a useful adjunct to endomyocardial biopsy for monitoring post-cardiac transplantation patients, and its use as a prospective diagnostic tool merits further study.

To generate a potent cell-mediated immune response, at least two signals are required by T cells. One is engagement of the T-cell receptor with peptide-bearing major histocompatibility complex molecules. The other signal can be delivered by various molecules on the antigen-presenting cell, such as B7-1 (CD80). Many tumor cells escape immune recognition by failing to express these costimulatory molecules. Transfection of the B7 gene into some murine tumor cells allows for immune recognition and subsequent rejection of the parental tumor. We have studied an alternative approach for the introduction of B7-1 onto the surface of tumor cells. This method involves purified glycosyl-phosphatidylinositol (GPI)-anchored proteins which can spontaneously incorporate their lipid tail into cell membranes. We have created and purified a GPI-anchored B7-1 molecule (called GPI-B7) which is able to bind its cognate ligand, CD28, and incorporate itself into tumor cell membranes after a short incubation. Tumor cells that have been reconstituted with GPI-B7 can provide the costimulatory signal needed to stimulate T cells. These findings suggest an approach for the introduction of new proteins onto cell membranes to create an effective tumor vaccine for potential use in human immunotherapy.

Heat-stable antigen (HSA/J11d/possibly homologous to CD24), a cell adhesion molecule capable of providing a co-stimulatory signal for T cell proliferation, is expressed on B cells, activated T cells, monocytes, granulocytes, Langerhans cells and thymocytes. Recent studies have demonstrated that co-stimulatory signals provided by cell adhesion molecules such as B7-1 play an essential role in generation of an anti-tumor immune response. To examine whether the co-stimulatory signal provided by HSA can induce an anti-tumor immune response, we have transfected HSA cDNA into the murine melanoma cell line K1735M2, and examined the ability of this transfected cell line to induce tumor-specific T cell responses. The results demonstrate that spleen cells from mice immunized with HSA-transfected K1735M2 cells showed enhanced T cell proliferation in a mixed lymphocyte tumor reaction (MLTR) assay and also demonstrated a significant anti-tumor cytotoxicity to the parent tumor cell (K1735M2). This anti-tumor cytolytic activity could be abrogated by pretreatment of effector cells with anti-mouse CD8 monoclonal antibody and complement. Under similar conditions, spleen cells from C3H mice immunized with vector-transfected K1735M2 cells neither actively proliferate in an MLTR assay, nor did they exert significant cytolytic activity against the respective tumor cells. In summary, our study demonstrated that HSA can provide a co-stimulatory signal for the T cell immune response against tumor cells in a murine model.

The absence of detectable levels of CSF-1 in op/op mice results in a marked deficiency of macrophage colony-stimulating activity (M-CSA) in both the unstimulated and postendotoxin sera of these animals. These deficiences are not secondary to the presence of an inhibitor of macrophage formation. In contrast, various organs, particularly the organs of endotoxin-treated op/op mice, released amounts of M-CSA comparable to those of normal mice. Similarly, mitogen-stimulated lymphoid cells from op/op mice released either similar or increased amounts of M-CSA compared to mitogen stimulated +/+ lymphoid cells. On the other hand, conditioned media from cultures of fibroblastoid cells obtained from primary and secondary cultures of op/op organs were nearly totally devoid of M-CSA. However, incubation of these op/op fibroblasts with endotoxin in vitro induced the easily and readily detectable levels of M-CSA. These data suggest that CSF-1 is most likely a major source of M-CSA in serum and postendotoxin serum, while its contribution to soluble M-CSA in other organs may be only partial. In addition, in specific circumstances, the induced release of other macrophage growth factors may partially compensate for CSF-1 deficiency. Furthermore, it appears that the generalized macrophage deficiency in op/op mice cannot be fully explained by the deficiency of soluble M-CSA (soluble CSF-1) and argues for an in vivo role of membrane-bound CSF-1. These data may be interpreted as supporting a model in which the regulation of CSF-1-dependent and CSF-1-independent macrophage production is carried out by partly unrelated mechanisms.

In vitro polyclonal activation of peripheral blood mononuclear cells (PBMCs) from 70% of the simian immunodeficiency virus (SIV) serum enzyme-linked, immunosorbent assay (ELISA)-negative sooty mangabeys leads to synthesis and release of low but significant and reproducible levels of SIV-reactive antibodies, as determined by ELISA and Western blot analysis. The predominant isotype of SIV-reactive antibodies in the pokeweed mitogen (PWM) supernatant fluids from serum ELISA-negative mangabeys is IgM, whereas the predominant isotype of SIV-reactive antibodies in seropositive mangabeys is IgG. Depletion of CD8+ cells led to a marked increase in the levels of SIV-reactive antibodies detected in supernatant fluids from PWM-induced cultures from the serum ELISA-negative mangabeys. No evidence for such SIV-reactive antibodies has been found, to date, in similar unfractionated or CD8+ T-cell-depleted PWM-induced PBMC cultures from uninfected macaques. Supernatant fluids from PWM cultures of PBMCs from a select group of serum ELISA-negative mangabeys, when concentrated five times, were shown to give a Western blot profile against SIV, similar to the profile seen with plasma from seropositive infected macaques and mangabeys. Evidence is presented to show that these serum ELISA-negative mangabeys are most likely latently infected with SIV. This evidence, which was obtained in samples from such ELISA-negative mangabeys, includes the detection of reverse transcriptase activity and the presence of SIV p27 in supernatant fluids of phytohemagglutinin-stimulated PBMCs in vitro. In addition, the data show the presence of CD8+ T cells that regulate SIV-specific Ig synthesis and show the detection of gag sequences by the polymerase chain reaction. Thus, the PWM assay described herein may provide a valuable additional tool for detection of lentivirus infection before or in the absence of seroconversion.

Normal FHCMs, or transformed cell lines derived from FHCMs, such as W1, even after induction of MHC antigens by pretreatment with IFN-gamma, failed to induce proliferation of allogeneic human PBMCs in vitro. To test the hypothesis that antigen-specific T-cell activation and proliferation require not only the binding of the TCR with its ligand, the MHC molecule, but also a second signal that involves the interaction of T-cell surface molecules with their natural ligands on the stimulating cells, a mAb against CD28 was used. Cocultures of allogeneic PBMCs with IFN-gamma-pretreated irradiated FHCMs or the W1 cell line in microtiter plates containing immobilized anti-CD28 mAb induced marked stimulator cells MHC class-II-specific proliferative responses. The W1 cell line and FHCMs failed to express detectable levels of the BB1/B7 molecule (the natural ligand for CD28) as determined by flow microfluorometry or mRNA levels coding for BB1/B7 as determined by RT-PCR. These data suggest that one of the probably reasons for the failure of MHC-expressing cardiac myocytes to induce allogeneic activation is the absence of costimulatory signals.

To test the hypothesis that CD8+ T cells inhibit viral replication at the level of cellular activation, an Epstein-Barr virus (EBV)-transformed cell line (FEc1) from a simian immunodeficiency virus (SIV)-seropositive sooty mangabey monkey was transfected with a human CD4 gene and shown to be replication-competent for HIV-1, HIV-2 and SIV. Utilizing a dual-chamber culture system, it was found that inhibition of viral replication can be mediated by a soluble factor. The FEc1 cell line was transiently transfected with an LTR-driven CAT reporter gene. It was found that autologous CD8+ T cells markedly inhibited CAT activity. Furthermore, co-transfection of the FEc1 cell line with an LTR-driven tat plasmid and LTR-CAT was able to quantitatively mitigate the suppressive effect. Thus, this inhibition appears to be directed at cellular mechanisms of viral transcription. Control transfections with an LTR-driven CAT plasmid with a mutation at the NFkB binding site yielded no CAT activity, suggesting that most viral replication as measured by CAT activity is dependent, to a large extent, upon cellularly derived NFkB binding proteins.

Osteopetrosis and the absence of colony-stimulating factor 1 (CSF-1) in op/op mice are associated with decreased cellularity of the bone marrow (to one tenth of the normal), a very significant reduction in the number of cells recovered from peritoneal, pleural, and alveolar lavages, moderate leukopenia, and a slight decrease in the number of cells per spleen and thymus. Furthermore, op/op mice possess deficiencies in the number of macrophages in various organs. These cells are apparently absent in the bone marrow, severely reduced (5%-15% of the normal number) in peritoneal and pleural cavities and in the lungs. In addition, a marked decrease in the frequency and total number of circulating monocytes is present (5% of the normal). The deficiency of macrophages is less severe in the liver, spleen, and thymus of op/op mice (approximately 30% of those seen in normal). There is a concomitant redistribution of macrophage progenitor cells (granulocyte-macrophage colony-forming units, CFU-GM) in op/op mice from the marrow to the spleen and liver, associated with an increased sensitivity to interleukin 3 (IL-3). Their total number is decreased at least threefold compared to control mice. Moreover, op/op mice have at least a fivefold reduction in the total number of day-11 spleen colony-forming units (CFU-S) associated with their redistribution to the spleen and liver. These data suggest that the macrophage system in op/op mice is reduced at all levels tested, that is, at the level of mature macrophages, the level of progenitors, and the level of stem cells, whereas the redistribution of progenitor and stem cells could be viewed as a secondary consequence of osteopetrosis. Furthermore, these data suggest that macrophage dependency in vivo on CSF-1 is limited and different in various organs. Particularly in the liver, spleen, and thymus, other growth factors may significantly compensate for CSF-1 deficiency. Based on the relative decrease in the number of CFU-GM in the op/op mice, it appears that the population size of these progenitors is less dependent on CSF-1 than the hematopoietic stem cell population size as evidenced by the day-11 CFU-S assay. The day-11 CFU-S population is severely reduced in op/op mice, suggesting a physiological involvement of CSF-1 in expanding its size. These data provide evidence that CSF-1, besides acting on the final and intermediate stages of macrophage maturation, may also play a role in early stages of hematopoiesis.

Studies of cultures and cloned T-cell lines from mononuclear cell infiltrates in cardiac biopsy specimens have provided a unique resource to study the cellular basis of human organ allograft rejection. Our laboratory has previously shown that biopsy specimens placed on autologous donor MHC-class-II-specific cloned T-cell lines from previous cardiac biopsies led to the isolation of cloned T-cell lines, which appeared to be functionally "antiidiotypic" in nature. Detailed functional analysis of such CD4+ individual antiidiotype-reactive cloned T-cell lines revealed that although some augmented the proliferative response of autologous idiotype-bearing cloned T-cell lines against donor stimulator cells, others markedly suppressed the proliferative response; thus, although each of these antiidiotype-like reactive cloned T-cell lines appears to specifically react with the same idiotype-bearing donor MHC-class-II-specific cloned T-cell line, they were functionally heterogeneous. Analysis of cytokines secreted by these individual clones showed that the antiidiotype-reactive cloned T-cell lines that suppressed the response of idiotype-bearing cells appear to secrete predominantly interferon gamma, whereas those antiidiotype-reactive cloned T-cell lines that augmented the response do not secrete interferon gamma but secrete interleukin-2, -4, and -6. These preliminary data suggest that differences in the predominant cytokines secreted by these individual antiidiotype-reactive cloned T-cell lines may account for their functional differences.

Normal human cardiac myocytes failed to induce proliferative responses of peripheral blood mononuclear cells from major histocompatibility complex (MHC)-disparate individuals. Induction of cell-surface MHC antigens on such myocytes before culture still failed to induce alloproliferative responses. Similar data were obtained with cocultures of a human cardiac myocyte (W1) cell line and allogeneic lymphocytes. Addition of interleukin (IL)-1, IL-2, or IL-1 plus IL-2 failed to reconstitute the alloproliferative response. Of interest was the observation that preincubation of primary cultures of myocytes or the W1 cell line (especially after pretreatment with interferon-gamma) with an autologous enriched population of adherent cells, containing monocytes/macrophages/B cells, markedly augmented the ability of these cells to induce alloproliferative responses. In addition, the specificity of the secondary response of T cells primed against myocytes or the W1 cell line (especially after pretreatment of the myocytes or the W1 cell line with interferon-gamma) appeared to reside at the MHC type of the adherent cells used in the primary phase. These data suggest that adherent cells most likely present donor myocyte-specific MHC or other proteins to allogeneic T cells in the context of the MHC molecules of the adherent cells. Such a finding provides evidence that the indirect pathway of host cellular sensitization may occur in vivo and play a role in chronic graft rejection.

In vitro culture of heart biopsy specimens from patients after transplantation in media containing recombinant human interleukin-2 led to the exudation of host mononuclear-cell infiltrates. Cloned T-cell lines were prepared from such infiltrates and studied for donor-specific mixed lymphocyte reaction and cytotoxic T-lymphocyte activity. Although most T-cell clones (greater than 50%) showed donor-specific reactivity, a small but distinct frequency (2% to 10%) of the cloned T-cell lines did not proliferate against donor or third-party stimulator cells. Of interest was our finding that addition of these non-donor-reactive cloned T-cell lines to autologous peripheral blood mononuclear cells markedly suppressed their donor-specific, but not third-party major histocompatibility complex, unrelated proliferative response and prevented the generation of donor, but not third-party, major histocompatibility complex unrelated cytotoxic T-lymphocyte function. The suppression was not secondary to lysis of donor stimulator cells, lysis of autologous donor-specific CD4+ lymphoblasts, or by selective consumption of interleukin-2. The suppression was mediated at the initiation of sensitization (precursor cell level). These suppressor cells expressed CD3, CD8, CD45RO, and the alpha, beta T-cell receptor, but not CD4 or CD56. These cloned T-cell lines will provide unique reagents to study the molecular basis by which these cells exert their regulatory function.

Mice that are mutant at the op locus have a severe deficiency of mononuclear phagocytes due to an inactivating mutation in the CSF-1 (macrophage colony-stimulating factor, M-CSF) gene. op/op mice are toothless, possessing skeletal abnormalities, a low body weight, and compromised fertility; they are osteopetrotic due to a deficiency of osteoclasts. The congenital osteopetrosis, toothless phenotype, osteoclast deficit, and the defects in splenic and femoral macrophages were corrected by routes of administration of human recombinant CSF-1 that maintained normal circulating CSF-1 concentrations. Early restoration of circulating CSF-1 was required for rescue of the toothless phenotype, but only partially restored body weight. In contrast, the deficiencies of pleural and peritoneal cavity macrophages and the reduced female fertility were not corrected by restoration of circulating CSF-1. These results suggest that although circulating CSF-1 is required for osteoclast and macrophage production, local synthesis and action of the growth factor are important for certain target cell populations.

Autoantibodies against the adenine nucleotide translocator (ANT), the branched chain alpha-ketoacid dehydrogenase (BCKD) complex proteins, and myosin have been implicated in the pathogenesis of human dilated cardiomyopathy (DCM). Cardiac tissue from patients with DCM and, for control purposes, cardiac tissue from patients with other forms of cardiomyopathy and from patients with no history of cardiac disease were stained with heterologous and ANT-, BCKD-, and myosin-specific affinity-purified sera from DCM patients. Data demonstrate that although anti-myosin stains tissues from both patients and normal controls, the ANT- and BCKD-specific heterologous and affinity-purified sera from DCM patients stain only cardiac tissues from DCM patients. Intense staining in patchy areas of cardiac tissue suggests that abnormal increased expression of these putative autoantigens occurs in discrete areas of cardiac myocytes. The reactivity of the antisera was organ specific and only seen in tissues from DCM patients. The organ and disease specificity of these findings suggests that such expression may play an important role in the pathogenesis of human DCM.

Techniques such as polyclonal B cell activation with pokeweed mitogen (PWM) and polymerase chain reaction (PCR) analysis have documented the existence of simian immunodeficiency virus (SIV)-and human immunodeficiency virus type 1-seronegative but infected humans and nonhuman primates. To establish whether blood from such seronegative but PWM- and PCR-positive monkeys can transmit infection, naive macaques were transfused with whole blood (n = 2) or cultured cells and supernatant fluid (n = 2) from two seronegative but PWM- and PCR-positive sooty mangabeys. After transfusion, three of the four recipients seroconverted, and peripheral blood mononuclear cells from all four recipients secreted SIV-reactive antibodies upon polyclonal activation in vitro and were SIV-positive by PCR that used highly specific gag primer pairs and probe. In addition, CD8+ cells from all four recipients markedly inhibited replication of SIV in autologous cells in vitro. These data suggest caution in the sole use of serologic tests for the detection of retroviral infection and document the ability of such blood samples to transmit infection.

Human cardiac myocytes do not express detectable levels of major histocompatibility complex (MHC) class II antigens and express low levels, if any, of MHC class I antigens. During rejection episodes, cardiac biopsies show massive increases of MHC antigens, which are thought to be induced by cytokines released by donor-sensitized recipient mononuclear cells. In efforts to determine the nature of the cytokines that induce MHC expression on cardiac myocytes, human fetal cardiac myocyte cultures were established. Interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma), interleukin (IL)-1, IL-2, IL-3, IL-4, and tumor necrosis factor (TNF)-alpha were added to these cultures and dose/kinetics of MHC class I/II induction quantitated. Data show that IFN-gamma induces both MHC class I and II expression, and all the other cytokines (except IL-2) induce only MHC class I but not class II. Cytokines used in combination showed that IFN-alpha with TNF-alpha was the only combination that induced MHC class II expression. Addition of immunosuppressive drugs such as cytoxan, azathioprine, cyclosporine-A, and FK-506, even when added at the initiation of the cultures, did not appreciably affect the ability of the appropriate cytokines to induce MHC expression by the myocytes in vitro.

Human cardiac myocytes undergo degeneration, cytolysis, and necrosis in a number of clinical disease conditions such as myocarditis, dilated cardiomyopathy, and during episodes of cardiac allograft rejection. The precise cellular, biochemical, and molecular mechanisms that lead to such abnormalities in myocytes have been difficult to investigate because at present it is not possible to obtain and maintain viable cell cultures of human adult cardiac myocytes in vitro. However, human fetal cardiac myocytes are relatively easy to maintain and culture in vitro, but their limited availability and growth, variability from one preparation to another, and varying degrees of contamination with endothelial and epithelial cell types have made it difficult to obtain reliable data on the effect of cardiotropic viruses and cardiotoxic drugs on such myocytes. These thoughts prompted us to attempt to derive a cell line of human cardiac origin. Highly enriched human fetal cardiac myocytes were transfected with the plasmids pSV2Neo and pRSVTAg and gave rise to a cell line (W1) which has been maintained in culture for 1 yr. Morphologic and phenotypic analyses of W1 cells by flow microfluorometry and immunoperoxidase techniques indicate that the W1 cell line shares many properties of human fetal cardiac myocytes, but appears not to react with specific antibodies known to react with markers unique to human endothelial, epithelial, skeletal muscle, and dendritic cells. These preliminary data suggest that the W1 cells may provide a unique source of an established cell line that shares many properties ascribed to human cardiac myocytes.

While normal cardiac tissue expresses low levels of MHC-class I, undetectable levels of MHC-class II antigens, and no mononuclear cell infiltrates, posttransplantation allogeneic donor cardiac tissue demonstrates dramatic increases of MHC-class I/class II expression coincident with the infiltration of the tissue with mononuclear cells. Results of this study demonstrate that the kinetics of MHC-class I/II antigen expression and the phenotype of mononuclear cell infiltrate are influenced, to a great degree, by the genetic H-2, intra-H-2 and non-H-2 incompatibility between donor and recipient strains of mice. Increases of MHC-class I precede class II expression in cells from donor cardiac tissue from completely allogeneic BALB/c, H-2-disparate B10.D2, B10.BR, and K, I-A and I-E-disparate B10.T (6R) strains of mice implanted in B10 recipients. In contrast, increase in the level of MHC-class II precedes MHC-class I increases in donor cardiac tissue from H-2-identical but non-H-2-incompatible A. By and the I-E + H-2D end-different B10.A(5R) donor tissue. The completely allogeneic, H-2-disparate or K, I-A, I-E-disparate donor cardiac tissue induced the infiltration of predominantly CD8+ T cells, whereas the non H-2 and I-E + H-2D end-different donor cardiac tissue induced the infiltration of predominantly CD4+ T cells. Finally, whereas bm1 donor cardiac tissue is rejected by B6 recipients by day 32, the (bm1 x bm12)F1 allografts are rejected by day 20, and both express MHC-class I antigens followed by MHC-class II antigens, and contain predominantly CD8+ T cells. In contrast, bm12 allografts are not rejected by B6 recipients, express chronic low levels of both MHC-class I and II antigens, and contain predominantly CD4+ T cells. Of interest is our preliminary finding that bm12 allografts placed in one ear of B6 recipients appear to modify the kinetics of MHC antigen expression and the predominant phenotype of mononuclear cell infiltrates in bm1 allografts placed in the opposite ear. Cumulatively, these data suggest that the type of genetic disparity between cardiac donor and recipient greatly influences the quantitative and qualitative host responses.

CD4+ T cells were cultured from posttransplant cardiac biopsies placed on irradiated feeder cells of autologous cloned donor major histocompatibility complex class II-specific T-cell lines cultured and grown from previous biopsies. Fourteen of the CD4+ T-cell cultures were expanded and cloned using the same feeder cells. Two of the 14 cloned T-cell lines were examined in detail for their ability to proliferate in vitro. Clones 7E4 and 8G2 proliferated (as determined by primed lymphocyte testing) only when cocultured with a series of distinct autologous cloned T-cell lines from previous biopsies that were specific for donor-specific HLA-DR3 and HLA-DR4, respectively. In addition, when HLA-DR-specific T-cell lines were established using recipient peripheral blood mononuclear cells and a series of HLA-DR-expressing homozygous typing cells, clone 7E4 only responded to the series of distinct HLA-DR3-specific autologous cloned T-cell lines but not to autologous HLA-DR2 and -DR4, and clone 8G2 responded to 3 of 8 distinct autologous HLA-DR4-specific T-cell lines, but not HLA-DR2-specific T-cell lines. These data demonstrate that cardiac biopsies contain CD4+ T cells of recipient origin which show anti-idiotype-like reactivity against T-cell receptors specific for donor-specific major histocompatibility complex class II molecules.

Osteopetrotic (op/op) mutant mice suffer from congenital osteopetrosis due to a severe deficiency of osteoclasts. Furthermore, the total number of mononuclear phagocytes is extremely low in affected mice. Serum, 11 tissues, and different cell and organ conditioned media from op/op mice were shown to be devoid of biologically active colony-stimulating factor 1 (CSF-1), whereas all of these preparations from littermate control +/+ and +/op mice contained the growth factor. The deficiency was specific for CSF-1 in that serum or conditioned media from op/op mice possessed elevated levels of at least three other macrophage growth factors. Partial correction of the op/op defect was observed following intraperitoneal implantation of diffusion chambers containing L929 cells, which in culture produce CSF-1 as their sole macrophage growth factor. No rearrangement of the CSF-1 gene in op/op mice was detected by Southern analysis. However, in contrast to control lung fibroblasts, which contained 4.6- and 2.3-kilobase CSF-1 mRNAs, only the 4.6-kilobase species was detected in op/op cells. An alteration in the CSF-1 gene is strongly implicated as the primary defect in op/op mice because they do not contain detectable CSF-1, their defect is correctable by administration of CSF-1, the op locus and the CSF-1 gene map within the same region of mouse chromosome 3, their CSF-1 mRNA biosynthesis is altered, and the op/op phenotype is consistent with the phenotype expected in a CSF-1 deficient mouse.

The measurement of cell-mediated immunity against the etiologic agent of human AIDS (HIV) in the non-human primate model of AIDS (simian immunodeficiency virus, SIV) has been difficult. In general, culture of peripheral blood mononuclear cells from HIV-1- and SIV-infected humans and monkeys, respectively, with purified inactivated HIV and SIV virus preparations has given inconsistent or negative proliferative responses. However, we describe herein an assay which consists of coculturing monocytes that have been pulsed with inactivated SIVsmm with nylon-wool-purified autologous T cells, leading to antigen-specific T-cell proliferation. The proliferative response, which predominantly occurs in CD4+ T cells, is major histocompatibility complex (MHC) class II-restricted and requires antigen processing. This assay will greatly facilitate the identification of the immunodominant epitopes recognized by T cells in sooty mangabeys, which are naturally infected but remain clinically asymptomatic, and in rhesus macaques, in which experimental infection leads to clinical symptomatology similar to human AIDS, eventually resulting in death.

Identification of human immunodeficiency virus type 1 (HIV-1)-infected individuals is of paramount importance for the control of the spread of AIDS worldwide. Currently, the vast majority of screening centers throughout the world rely on serological techniques. As such, clinically asymptomatic but HIV-infected, seronegative individuals are rarely identified. In this report we show that 18% (30/165) of seronegative individuals who were considered to be a unique cohort of patients at high risk for HIV infection had circulating B cells that, upon in vitro polyclonal activation with pokeweed mitogen, produced antibodies reactive with HIV. Furthermore, polymerase chain reaction analysis of DNA obtained from aliquots of the peripheral blood mononuclear cells from these seronegative but pokeweed mitogen assay-positive individuals tested revealed the presence of HIV-specific sequences in a significant number of samples. In addition, depletion of CD8+ T cells from peripheral blood mononuclear cells of HIV-1-seronegative individuals prior to in vitro culture with pokeweed mitogen resulted in increased sensitivity for detecting HIV-reactive antibodies. This assay has obvious epidemiological implications, especially in the case of high-risk groups, and also provides a simple technique to enhance detection of HIV-infected individuals. Of further interest is the determination of the mechanisms related to the lack of HIV-specific antibodies in the serum of these infected individuals.

The histologic diagnosis of active myocarditis is frequently difficult to establish. A nonhistologic marker of immune activation would be clinically useful in identifying cases of immune-mediated myocarditis. A viral etiology with subsequent autoimmunity to cardiac antigens has been implicated in human myocarditis. Because autoimmunity and viral disease are commonly associated with increased expression of major histocompatibility complex (MHC) antigens on targeted tissue, we examined endomyocardial biopsy samples from patients with active myocarditis for abnormal levels of MHC antigen expression. Thirteen patients with active myocarditis and eight control patients with other well-defined cardiac diagnoses (coronary disease, amyloidosis or neoplasm) were studied. A sensitive radioimmunoassay was developed that utilized monoclonal antibodies to human MHC class I and class II antigens in order to quantitate the expression of both of these antigens within each biopsy. Abnormal MHC class I and class II antigen expression was present in 11 of 13 myocarditis specimens and 1 of 8 control samples (specificity 88%, sensitivity 84.6%). Active myocarditis samples had approximately a 10-fold increase in MHC class I and class II expression. Immunoperoxidase staining localized abnormal MHC expression primarily within microvascular endothelium and along myocyte surfaces (11 of 13). This study is the first to demonstrate a marked increase in major histocompatibility complex antigen expression within the myocardium of patients with active myocarditis. The identification of abnormal histocompatibility antigen expression within an endomyocardial biopsy may prove a useful adjunct to the histologic diagnosis of myocarditis.

Several investigators have demonstrated the ability of CD8+ T cells from HIV-1 infected humans and SIV infected rhesus macaques to inhibit viral replication in vitro. In this report we show that CD8+ cells from naturally SIV infected sooty mangabeys also have the ability to inhibit viral replication in vitro. In addition, initial experiments which seek to elucidate the mechanism and antigen specificity of CD8-mediated suppression are described.

Monocytes from SIV/SMM infected sooty mangabeys and rhesus macaques were incubated in vitro with live SIV/SMM. The reverse transcriptase (RT) activity in the supernatant fluids of the monocyte cultures of the former species was higher than the RT activity in the latter species. No differences were found in the supernatant fluid of similar cultures of CD4+ T cells from both these species. Autologous (but not allogeneic) CD8+ T cells from SIV infected mangabeys and rhesus macaques inhibited SIV replication in vitro. The suppression appeared more marked in monocytes from the mangabey species. These in vitro differences may relate to the clinically asymptomatic state of the sooty mangabeys and the disease-susceptible state of the rhesus macaques.

Initial studies have revealed subtle differences in the T cell proliferative response to whole SIV antigen in the peripheral blood mononuclear cells (PBMC) from sooty mangabeys and rhesus macaques. Preliminary findings utilizing the cellular Western blot assay are described.

Conflicting reports on the immune responsiveness of patients with subacute sclerosing panencephalitis (SSPE) have been reported. This report shows that the leucocytes from four SSPE patients exhibited strong sensitivity to both measles and SSPE virus preparations as measured by the macrophage migration inhibition test, mixed lymphocyte virus infected cell culture test, and the lymphotoxin assay. Earlier suggestions that a factor may be operating to suppress cellular reactivity are confirmed by the demonstration that the response of lymphocytes from SSPE patients could be blocked by the addition of SSPE spinal fluid or plasma. It was determined that the blocking factor was stable at -20 degrees C, heat labile at 56 degrees C for 30 minutes, trypsin and neuraminadase sensitive, and had a mol wt greater than 150,000 as determined by Sephadex G-200 gel chromatography. The blocking factor appeared to be specific for SSPE virus and did not block the response of lymphocytes to nonspecific mitogenic agents and other viral and bacterial agents.

Mononuclear cells (10--20 X 10(6)) obtained from human peripheral blood by a standard Ficoll-Hypaque technique were suspended in RPMI 1640 media at 4 degrees C containing 10% foetal calf serum and 7-5% dimethyl sulphoxide (DMSO). Two-millilitre aliquots were cooled at -1 degree C/min in a Cryoson BV-4 programmed freezing system to -30 degrees C, then -5 degrees C/min to -80 degrees C and stored in liquid nitrogen vapor. On the day of testing, cell suspensions were thawed rapidly in a 37 degree C water bath. DMSO was diluted slowly out of the sample and cells resuspended in fresh RPMI 1640. It was found that frozen stored human lymphocytes (FSHL) demonstrated all the characteristics of fresh unfrozen cells. These included their ability to form spontaneous rosettes with sheep erythrocytes ('E' rosettes) and sheep erythrocyte--antibody--complement rosettes ('EAC' rosettes). The presence of surface immunoglobulins and Fc receptors were shown by membrane immunofluorescence to be comparable. In addition, the results show that FSHL respond to mitogens, specific antigens; act as both stimulators and responders in the mixed lymphocyte culture reaction; and exhibit cell-mediated lymphocytotoxicity following in vitro sensitization, or against antibody-coated target cells.

An attempt was made to prepare a specific heterologous rabbit antimouse T cell antiserum (anti-MTLA) by absorbing rabbit antimouse thymocyte globulin (ATG) with spleen cells from BALB/c TXBM mice. Cytotoxicity data showed that whereas ATG was cytotoxic to both T and B cells, anti-MTLA was highly cytotoxic to only T cells. Whereas spleen cells treated with ATG and complement (C) failed to respond in all assays studied, spleen cells treated with anti-MTLA and C: (1) responded to the B cell mitogens but failed to respond to the T cell mitogens; (2) were able to stimulate allogeneic spleen cells but failed to respond to mixed lymphocyte culture (3) failed to act as T killer cells in the CML reaction but retained their ability to kill antibody-coated target cells; and (4) did not cause a graft-versus-host reaction when injected in allogeneic mice and increased their survival significantly. Furthermore, anti-MTLA was just as immunosuppressive in vivo as ATG in its ability to suppress the immune response to sheep red blood cells and prolong skin allograft survival. Anti-MTLA was found to be different in specificities from anti-theta serum by several points: (1) it was cytotoxic for T cells from both theta-C3H and theta-AKR mice; (2) it was highly immunosuppressive in vivo when compared to anti-theta serum; (3) absorption of anti-MTLA with mouse brain did not decrease the immunosuppressive activity; and (4) rabbit antimouse brain antiserum failed to show any immunosuppressive activity. These data indicate that anti-MTLA is a specific antiserum against a unique marker on T cells distinct from the theta marker.