C-kit+ FcR+ myelocytes are increased in cancer and prevent the proliferation of fully cytolytic T cells in the presence of immune serum

Immunogenic cancers induce both IgG antibodies and CD8(+) cytotoxic T lymphocytes (CTL). Rejection of almost all immunogenic tumors depends ultimately on CTL. When tumors grow progressively, IgG continues to be produced but CTL may no longer be demonstrable. Using syngeneic mixed lymphocyte tumor cell cultures, we found that proliferation of fully activated proliferating CTL is prevented by a small subpopulation of immature myeloid c-kit(+) FcR(+) cells, for convenience referred to as "barrier cells". Both, FcR on barrier cells and IgG linked to TGF-beta (IgG-TGF-beta) present in immune serum, are obligatory for barrier cells to prevent proliferation of CTL, suggesting that IgG-TGF-beta binds FcR to activate suppression. Growing tumors increase barrier cells in the spleen. Interfering with the cells or molecules essential for barrier cells to prevent proliferation of CTL may enhance tumor and other CD8(+) CTL-mediated immunity.     

Regulation of human cytotoxic lymphocyte responses. I. Non-cytolytic suppression mediated by alloantigen-activated cells.

Alloantigen sensitized human lymphocytes obtained from a 2-3 day mixed lymphocyte culture (MLC) suppressed the in vitro generation of alloreactive cytotoxic lymphocytes (CTL). The inhibition of CTL responses was demonstrated in MLC after both 1 day and 14 days' allosensitization. The suppressor cells were nylon wool non-adherent, lacked Fc receptors and adhered to histamine columns. The MLC-activated suppressor cell population had an associated very low and transient cytotoxic response directed against the allogeneic sensitizing cell. Several procedures were used to dissociate this activity from suppressor cell function: (1) donors were preselected which showed minimal cross-killing between allogeneic stimulating cells, (2) suppressor cultures were added to the test cultures prior to the development of maximal CTL activity, (3) suppressor cultures were irradiated preventing cell proliferation associated with differentiating CTL. Lastly, by increasing stimulating antigen concentration suppressor cell activity was increased rather than being competitively diminished, which would be predicted if suppression was occurring through a cytolytic or inactivation of stimulating antigen. It was therefore concluded that alloantigen stimulation in MLC activates a non-cytolytic regulatory cell population which is capable of inhibiting CTL responses to third-party allogeneic lymphocytes.     

Polyclonal Antibody Secretion Induced in Human Mixed Lymphocyte Cultures

Antibody secreting B cells were measured as plaque forming cells (PFC) in a hemolysis-in-gel assay using fluorescein isothiocyanate (FITC) coupled SRBC as targets or protein A coupled SRBC as targets and developing antisera. Peak antibody secretion occurred on day 5 and the highest number of PFC was seen when mitomycin treated stimulator cells: responder cells were used in a ratio of 1:4. The number of PFC in MLC was not correlated to the DNA synthetic response. Antibody secretion in MLC was found to be of IgM, IgG and IgA classes. Significant numbers of PFC in MLC from blood lymphocytes were detected with the protein A technique, but not using FITC-SRBC targets. Compared to spleen cells, fewer PFC were stimulated in blood lymphocytes. B cells alone, enriched by rosetting of T cells, did not respond by antibody secretion or DNA synthesis in MLC. When Lipopolysaccharide (LPS) was added to MLC an additional effect was seen on the number of PFC which may indicate that distinct B cell subpopulations are activated in MLC by LPS.     

Production and Main Characteristics of a Fetal Calf Serum-specific Cell Line that Induces T and B Cell Differentiation

Spleen cells from B6 mice injected with fetal calf serum (FCS) could be kept proliferating as a continuous cell line in vitro provided they were culture in the presence of irradiated syngeneic spleen cells and FCS. Cells in this cell line showed a strong proliferative response when stimulated with concanavalin A (Con A), and they were able to mediate the following functions: (1)they helped the generation of alloantigen-specific cytotoxic T lymphocytes (CTL) from thymocyte-spleen cell mixed lymphocyte cultures (MLC), (2)they induced the generation of CTL from normal syngeneic spleen cells in the absence of allogeneic stimulator cells, and (3)they induced normal spleen cells to differentiate into anti-sheep erythrocyte (SRBC) plaque-forming cells (PFC), in the absence of SRBC in the cultures. The use of this cell line (called line 12) may thus provide an interesting approach for the study of cellular and molecular requirements for cell-cell interactions and for the differentiation of T and B effector functions.     

Mitogenic Responsiveness of Human T-Lymphocyte Subpopulations: Regulation by Suppressive Fc-Receptor-bearing T Cells and Influence of Fractionation Procedures

The proliferative response induced by leucoagglutinin (La) in different subpopulations of human T lymphocytes was studied. Subpopulations enriched in cells with either high- or low-avidity receptors for sheep erythrocytes (SRBC) (EH +, EL +) were prepared by sequential E-rosetting. In addition, T lymphocytes prepared by E-rosetting under optimal conditions (E + TOT) were fractionated on wheat germ agglutin (WGA)-Sepharose columns, rendering fractions enriched in lymphocytes with either low- or high-avidity receptors for WGA. The T lymphocytes were found to comprise at least three functionally distinct subpopulations, differing with respect to mitogen responsiveness. Cells characterized by high-avidity receptors for WGA and SRBC were highly responsive to La stimulation, regardless of the method used for purification. In contrast, cells with low-avidity receptors for WGA and probably also for SRBC but lacking Fc receptors for IgG responded only marginally but were conditioned to respond when subjected to E-rosetting under optimal conditions. This response was suppressed by lymphocytes with Fc receptors for IgG, which probably also had low-avidity receptors for WGA and SRBC. The lymphocytes with high-avidity receptors for WGA ans SRBC did not appear to be susceptible to suppression by Fc gamma + cells.     

Polyclonal Antibody Secretion Induced in Human Mixed Lymphocyte Cultures

In mixed leucocyte culture (MLC), using human spleen cells or thoracic duct lymphocytes, antibody secretion was induced, measured as plaque-forming cells (PFC) in a haemolysis-in-gel assay with fluorescein isothiocyanate (FITC)-coupled sheep erythrocytes (SRBC) as targets. Peak antibody secretion was seen on day 5. Using protein-A-coupled SRBC as targets and developing antisera, antibody secretion in MLC was found to be of IgM, IgG and IgA type. There was no correlation between the number of PFC against FITC-SRBC in MLC and DNA synthesis. Supernatants from MLC failed to induce antibody secretion.     

IgG2b-dependent down regulation of the LPS-induced PFC-response and its blockade by Fc gamma 2bR protein

Fc gamma receptor (Fc gamma R)-dependent immunoregulation by murine heat-aggregated (HAgg) IgG subclasses on the bacterial lipopolysaccharide (LPS)-induced plaque forming cell (PFC) response to trinitrophenylated sheep red blood cell (TNP-SRBC) antigen and the competitive effect by Fc gamma 2bR-protein on the down regulation by HAgg-IgG2b were studied in murine T-cell-deprived spleen cell cultures. HAgg-IgG1 and HAgg-IgG3 enhanced the PFC response, but HAgg-IgG2b strongly suppressed the LPS-induced PFC response. HAgg-IgG1 could not compete with the suppressive effect of HAgg-IgG2b. The HAgg-IgG2b seemed to act on both macrophages (M phi) and B-cells, because the cell cultures that had been reconstituted with HAgg-IgG2b-pretreated M phi and untreated B-cells and vice versa showed poor PFC responses. The suppression induced by HAgg-IgG2b on the LPS-induced PFC response in the T-cell-deprived cultures was abolished by the addition of phospholipase C (PLC)-treated Fc gamma 2bR protein at the early stage of the culture. The mechanisms by which HAgg-IgG2b suppress the LPS-induced PFC response and PLC-treated Fc gamma 2bR protein restores this response were discussed.     

Regulation of human cytotoxic lymphocyte responses. II. Suppression by a soluble factor produced by primed lymphocytes.

A soluble supernatant factor is elaborated from in vitro primed human allogeneic lymphocytes which suppresses the development of alloreactive cytotoxic lymphocytes (CTL). In contrast, supernatants obtained from primary mixed lymphocyte cultures (MLC) and primed autologous cultures were unable to suppress CTL activation, indicating that antigen restimulation was required to elicit the factor. The suppressor factor (SF) functioned in a dose-dependent manner. When the SF was added 24 or 48 hr after MLC initiation it was ineffective. However, adding the SF at culture initiation significantly reduced CTL activity, suggesting that suppression occurs either during antigen recognition or early in the CTL differentiation pathway. The SF did not function by altering the kinetics of the CTL response. Preincubation experiments showed that the SF operates by partially inactivating both MLC responder and stimulator cell populations.     

Surface Markers on Human B and T Lymphocytes

The distribution of surface immunoglobulin and receptors for Fc, C3, and sheep erythrocytes (SRBC) on resting and blast-transformed peripheral lymphocytes was investigated. The following conclusions were reached. [1] SRBC receptors were retained on all blast-transformed T lymphocytes. No such receptors were found on normal or neuraminidase-treated B lymphocytes. [2] Receptors for Fc and C3 were found to be expressed on the same resting lymphocytes, which formed a subpopulation that did not entirely overlap with surface immunoglobulin-positive B cells. Owing to this and the fact that Fc/C3 receptors were lacking on almost all B blast cells, as well as for other reasons elaborated in the text, it is argued that caution must be taken when these receptors are used as B-cell markers. [3] A comparison among C3 indicator cells prepared with human or mouse complement showed that these detected the same lymphocyte subpopulatiom [4] B blast cells, induced by 72-hr pokeweed mitogen cultures, were found to carry detectable amounts of surface immunoglobulin. [5] Phagocytic leukocytes were found to be conveniently detected by scoring the number of cells with internal C3 indicator cells after osmotic lysis of externally bound indicator cells.     

Cytotoxic T cell clone-specific monoclonal antibodies used to select clonotypic antigen-specific cytotoxic T cells

Two rat monoclonal antibodies (mAb) have been produced which recognize a clone-specific determinant on the alloreactive cytotoxic T lymphocyte (CTL) clone 3 F9. CTL clone 3F9 of BALB/c origin is specific for H-2Db and can be grown by weekly restimulation with irradiated stimulator spleen cells expressing H-2Db in the presence of interleukin 2. Two mAb against T cell clone 3F9, 44-22-1(IgG2a) and 46-6 B5(IgM), have been proven to be clone specific: they inhibit cytotoxic activity of 3F9 only and bind specifically to 3 F9 when compared in a panel of different CTL clones, or cells from different mixed lymphocyte cultures (MLC), BALB/c thymus and spleen cells. The mAb 44-22-1 has been used to sort cells from a primary MLC BALB/c anti-H-2Db by fluorescence-activated cell sorter (FACS) to select CTL expressing 3 F9 clonotype-specific determinants. The lymphocytes reactive with 44-22-1 represent a minor subpopulation of the CTL of the primary MLC. The specific alloreactive cytotoxicity of unsorted lymphocytes of the bulk primary MLC could not be inhibited by the mAb 44-22-1 and 46-6 B5 whereas the sorted 3 F9 clonotype-positive cultures could be inhibited very effectively. All the CTL clones derived from the FACS-sorted clonotype-positive culture show all the same properties and are identical with clone 3 F9 with respect to antigen-specific cytotoxicity, inhibition of cytotoxicity by the mAb and surface markers.     

Mesenchymal stem cells inhibit the expression of CD25 (interleukin-2 receptor) and CD38 on phytohaemagglutinin-activated lymphocytes

Mesenchymal stem cells (MSC) are immunomodulatory and inhibit lymphocyte proliferation. We studied surface expression of lymphocyte activation markers and secreted cytokines, when lymphocytes were activated in the presence of MSC. MSC suppressed the proliferation of phytohaemagglutinin (PHA)-stimulated CD3+, CD4+ and CD8+ lymphocytes. MSC significantly reduced the expression of activation markers CD25, CD38 and CD69 on PHA-stimulated lymphocytes. Mixed lymphocyte culture (MLC) supernatants containing MSC suppressed proliferation of MLC and PHA-stimulated lymphocytes dose-dependently. MSC secrete osteoprotegerin (OPG), but not hepatocyte growth factor (HGF) or transforming growth factor-beta (TGF-beta). Stromal-cell-derived factor-1 (SDF-1) is not expressed on the cell surface. A recent report suggested that T-cell suppression by MSC is mediated by HGF and TGF-beta. MSC suppression was not restored by the addition of neutralizing antibodies against SDF-1, OPG, HGF or TGF-beta, alone or in combination. Addition of guanosine to PHA-stimulated lymphocyte cultures containing MSC did not affect lymphocyte proliferation. The immunosuppressive effects of cyclosporine and MSC did not interfere, when present in the cultures of PHA-activated lymphocytes. In summary, human MSC suppress proliferation of both CD4+ and CD8+ lymphocyte and decrease the expression of activation markers.     

Induction of alloreactive immunosuppression by 1,4-bis [( 2-aminoethyl)amino]-5,8-dihydroxy-9,10-anthracenedione dihydrochloride (CL 232,468)

1,4-bis[2-aminoethyl)amino]-5,8-dihydroxy-9,10-anthracenedione (AEAD) has been investigated for its potential immunosuppressive effect on cell-mediated immune responses. Addition of the compound to mixed lymphocyte cultures (MLC) not only significantly inhibited these cells from responding to alloantigens but also prevented the induction of cytolytic T lymphocytes (CTL). A structurally related compound, mitoxantrone, was also found to be active in inhibiting CTL induction. AEAD had to be present during the first 3 days of a 5-day MLC in order to produce a significant effect and it had no effect on those CTL already generated, suggesting that it acted upon induction of CTL rather than the effector phase. Lymphocytes from mice treated with the compound were incapable of responding to alloantigens in vitro and the effect was dose- and time-dependent. Furthermore, lymphocytes from treated mice were found to inhibit CTL generation from normal mouse lymphocytes, indicating that a suppressor cell population might be induced in the spleens of animals treated with the compound. The present findings clearly demonstrate that AEAD is a compound with potent immunosuppressive activity on alloreactive immune responses.     

Cross-linking of Fc(gamma)-receptor on monocytes inhibits hepatitis C virus-specific cytotoxic T-lymphocyte induction in vitro.

In hepatitis C virus (HCV) infection, immune complex (IC)-type virus particles are frequently observed in circulation. The IC leads to cross-linking of Fcgamma receptors (FcgammaR) on monocytes and exerts immunoinhibitory function. To test the roles of IC in HCV-specific cytotoxic T lymphocyte (CTL) induction, we generated HCV CTL from peripheral blood mononuclear cells of chronic hepatitis C patients with or without HCV-IC- or immunoglobulin G (IgG)-coated culture plates and compared their lytic activities. HCV-IC or adherent IgG, which induces FcgammaR cross-linking, significantly reduced CTL activity. Expression of B7-1 on monocytes decreased on adherent IgG. In addition, tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1) production increased from cells on adherent IgG and their mRNA expression in monocytes was enhanced. Anti-TNF-alpha antibody during induction on adherent IgG inhibited lysis; however, anti-TGF-beta completely reversed its inhibitory effect. These results demonstrated that HCV-IC or adherent IgG impaired HCV-CTL induction in vitro. The FcgammaR-mediated CTL suppression occurred via decreased expression of monocyte B7-1 and/or enhanced production of TGF-beta1.     

The proliferative capacity of human T lymphocyte subpopulations in a continuous culture system.

The continuous growth of subpopulations of human T lymphocytes was investigated using a culture system containing conditioning factors from mitogen-stimulated lymphocytes. Cultures of purified peripheral blood lymphocytes rapidly became enriched for T lymphocytes, detected as sheep erythrocyte rosette-forming cells, and could be maintained for up to a month in an actively growing state. Subpopulations of T lymphocytes bearing Fc receptors for IgM(T.M) or IgG(T.G) could not be detected in these cultures unless the cells were first washed and cultured for 3 days in medium devoid of conditioning factors. During this second culture step, many of the cells reverted from a large, blast-like state to a small lymphocyte morphology and proportion of the T lymphocytes re-expressed Fc receptors. The proportions of T.M and T.G lymphocytes so detected remained constant throughout the continuous culture period indicating that the system permitted the proliferation of all T lymphocytes. Fractionation studies supported this conclusion by demonstrating that purified T, T.M and T.G but not B lymphocyte populations proliferated when cultured in the presence of conditioned medium. The majority of cells in cultures of purified T.M lymphocytes re-expressed IgM Fc receptors following reculture in unconditioned medium. However, the re-expression of IgG Fc receptors by cultured T.G lymphocytes could not be achieved.     

Delineation of IgM-receptor bearing human T and B lymphocytes using a direct plaque forming cell (PFC) assay.

Based on the observation that binding of IgM cytophilic antibodies to lymphocytes is temperature dependent, a direct plaque forming cell (PFC) assay was developed to detect IgM-receptor bearing human peripheral blood T and B lymphocytes. Lymphocytes were passively sensitized with IgM anti-SRBC molecules at 4 degrees C, added to SRBC monolayers then incubated at 37 degrees C with guinea pig complement to develop the plaques. The PFC assay has methodological advantages over rosetting methods which demonstrate IgM receptors, and under certain conditions is more sensitive than these rosette techniques. A mean of 17% of freshly isolated uncultured lymphocytes, enriched for B cells, formed direct plaques while a mean of 3% of T-enriched preparations formed direct plaques. However, if the lymphocytes were preincubated with vibrio cholerae neuraminidase (VCN) these figures increased to 46% and 35% respectively. The specificity of plaque formation by VCN-treated lymphocytes was established. SRBC sensitized with a F(ab')2 preparation of an IgG anti-SRBC reagent failed to bind to VCN-treated lymphocytes, inclusion of IgM, but not other Ig molecules in the test medium, inhibited plaque formation, and, most important, plaque formation by T and B cells was inhibited by F(c)5 mu but not by Fab mu fragments. These results indicate that T and B lymphocytes express IgM-class specific membrane receptors, that these receptors may be hidden on normal lymphocytes but are revealed by treatment with VCN and that the IgM receptor on VCN-treated lymphocytes is F(c)mu specific. These findings are discussed briefly with regard to other and partly contradictory data obtained after overnight in vitro lymphocyte culture. As demonstrated by direct PFC assay, the B cell IgM receptor is trypsin sensitive.     

Mucosal homeostasis: role of interleukins, isotype-specific factors and contrasuppression in the IgA response

Oral ingestion of antigen elicits immune responses at mucosal sites where humoral immunity is largely due to antibodies of the IgA isotype. This is often accompanied by suppression of systemic responses to the same antigen, a state termed oral tolerance. This IgA response is regulated by interactions between T cell subsets found at IgA inductive tissues, i.e., the gut-associated lymphoreticular tissue (GALT) or Peyer's patches (PP). PP T helper (Th) cells support IgA responses, and interleukins 5 (IL-5) and IL-6 can augment secretion of this isotype. Subsets of Th cells may also express Fc receptors for IgA (Fc alpha R) and secrete Fc alpha R as an IgA-binding factor (IBF alpha). Membrane-derived Fc alpha R is a glycoprotein of 38,000 M.W. and this molecule induces selective increases in IgA secreting cells (as determined by the ELISPOT assay) in PP B cell cultures. Fc alpha R+ T cell lines have been shown to secrete IBF alpha as well as IL-5 both of which promote IgA synthesis. Recombinant IL-5 (rIL-5) and rIL-6 induce IgA synthesis mainly by PP B cell blasts, and principally act on surface IgA-positive (sIgA+) B cells for these responses. Another form of mucosal regulation is provided by T contrasuppressor (Tcs) cells, which abrogate oral tolerance when adoptively transferred to mice and restore systemic responsiveness to the antigen sheep erythrocyte (SRBC). Tcs cells from mice systemically primed with SRBC support IgM and IgG subclass responses, while Tcs cells from orally primed mice support IgM, IgG subclass and IgA anti-SRBC responses. These Tcs cells are CD3+, CD4-, 8- and are antigen-specific. These regulatory cells may use the gamma-delta (gamma-delta) form of T cell receptor for antigen recognition.     

Antibody synthesis by chicken spleen cells in vitro. I. Requirements of B cells at various stages after immunization for T cells,macrophages and antigen

The requirements for T cells, macrophages and antigen during the induction of in vitro antibody responses were ascertained with chicken spleen cells obtained at various times after immunization with sheep red blood cells (SRBC). The IgM plaque-forming cell (PFC) response was T cell independent exclusively in cultures initiated 3 days after priming, but macrophage dependent at all time intervals tested. In cultures started 4 to 10 days after priming the IgG response was both T cell and macrophage independent and PFC numbers remained at a high plateau level throughout the culture period. In contrast, IgG responses initiated more than 15 days after priming showed a reversal to complete T cell and macrophage dependence and were characterized by a sharp increase in PFC numbers between days 2 and 4 of culture. Formaldehyde-fixed SRBC were immunogenic for IgG PFC 4 to 10 days after priming but failed to stimulate later IgG memory and all IgM responses. Contrasting antigen dose requirements for IgM and IgG responses were found in cultures initiated at various periods after priming. The results suggest that direct contact with fixed antigen was sufficient to maintain IgG antibody synthesizing PFC in vitro, while native antigen and cell co-operation were required for late secondary IgG and all IgM responses. These results are interpreted in terms of separate pathways of differentiation for IgM and IgG antibody-producing cells. A distinct 3rd day stage of T cell-independent but macrophage-dependent responsiveness for both classes of antibody was also defined.     

Fc Receptor-Bearing Lymphoid Cells in the Chicken I. Characterization of the Fc(IgG) Receptor

The receptor for Fc(IgG) on chicken lymphoid cells was investigated by EA rosette techniques using sheep erythrocytes sensitized with a subagglutinating dose of anti-sheep erythrocyte (SRBC) chicken serum. Chicken lymphocytes did not form rosettes with SRBC coated with rabbit antibody, and human and mouse lymphocytes did not bind SRBC sensitized with chicken antibody. Only avian sera were effective in blocking the Fc receptor. Similarities between chicken and mammalian Fc receptors were demonstrated as both are pronase sensitive, trypsin resistant, and are distinct from surface immunoglobulin. Fc receptors were also distinguished from avian bursa-and thymus-specific antigens. Additional Fc receptor-bearing cells were revealed in bursa, spleen and bone marrow lymphocytes after neura-minidase treatment.     

Isolation and characterization of human fetal macrophages from placenta

Human fetal macrophages expressing class II major histocompatibility complex (MHC) antigens have been isolated from the stroma of the chorionic plate of term placentas, using enzymatic digestion procedures, and enriched by Percoll density centrifugation. These cells are adherent, phagocytic and express Fc receptors for IgG. By rosetting with bovine erythrocytes coated with IgG, they can be enriched to 77-95% purity. Placental macrophages isolated in this way stimulate the proliferation of lymphocytes from unrelated donors in mixed-cell cultures, and act as accessory cells in oxidative mitogenesis. In a family study, placental macrophages stimulated proliferation of maternal and paternal lymphocytes but there was no evidence for either priming to, or suppression by, the fetal cells when the responses of lymphocytes from the mother and her HLA identical twin were compared. The possibility that these cells can protect the fetus from infection and/or stimulate the production of maternal anti-fetal HLA-antibodies is discussed.     

Studies of the presence of membrane receptors for complement,IgG and the sheep erythrocyte rosetting capacity on the same human lymphocytes

Normal peripheral blood lymphocytes were tested by a mixed rosette method, employing different sized erythrocytes as indicators to identify lymphocytes simultaneously possessing membrane markers found commonly on B and T cells. Only small populations of these lymphocytes were detected regulary in normal lymphocyte preparations. One type of lymphocyte (ranged from 0.5%-8%) was shown to possess the following markers: receptors for human and rabbit IgG, receptors for the third complement component C3b and C3b inactivator-cleaved C3b (C3d), and the capacity to rosette spontaneously with uncoated sheep erythrocytes (SRBC). Another lymphocyte cell type was shown to possess both SRBC and IgG receptors but lack membrane immunoglobulins and complement receptors. This population was detected in lymphocyte preparations depleted of complement receptor cells, and an increased number of these cells was found in rosette preparations incubated with human serum. The possible presence of some lymphocytes possessing both complement and SRBC receptors and lacking other markers was considered. The possibility that these small populations of human lymphocytes are sub-populations of T cells with certain cytotoxic function is postulated.