An unappreciated role for neutrophil-DC hybrids in immunity to invasive fungal infections.

Neutrophils are classically defined as terminally differentiated, short-lived cells; however, neutrophils can be long-lived with phenotypic plasticity. During inflammation, a subset of neutrophils transdifferentiate into a population called neutrophil-DC hybrids (PMN-DCs) having properties of both neutrophils and dendritic cells. While these cells ubiquitously appear during inflammation, the role of PMN-DCs in disease remains poorly understood. We observed the differentiation of PMN-DCs in pre-clinical murine models of fungal infection: blastomycosis, aspergillosis and candidiasis. Using reporter strains of fungal viability, we found that PMN-DCs associate with fungal cells and kill them more efficiently than undifferentiated canonical neutrophils. During pulmonary blastomycosis, PMN-DCs comprised less than 1% of leukocytes yet contributed up to 15% of the fungal killing. PMN-DCs displayed higher expression of pattern recognition receptors, greater phagocytosis, and heightened production of reactive oxygen species compared to canonical neutrophils. PMN-DCs also displayed prominent NETosis. To further study PMN-DC function, we exploited a granulocyte/macrophage progenitor (GMP) cell line, generated PMN-DCs to over 90% purity, and used them for adoptive transfer and antigen presentation studies. Adoptively transferred PMN-DCs from the GMP line enhanced protection against systemic infection in vivo. PMN-DCs pulsed with antigen activated fungal calnexin-specific transgenic T cells in vitro and in vivo, promoting the production of interferon-γ and interleukin-17 in these CD4+ T cells. Through direct fungal killing and induction of adaptive immunity, PMN-DCs are potent effectors of antifungal immunity and thereby represent innovative cell therapeutic targets in treating life-threatening fungal infections.

Dendritic-tumor cell hybrids induce tumor-specific immune responses more effectively than the simple mixture of dendritic and tumor cells.

BACKGROUND AIMS: Dendritic cell (DC)-tumor cell hybrids have been used clinically in cancer immunotherapy, but their advantage over the simple mixture of tumor cells and DCs is still a matter of controversy. In this study, we compared DC-tumor cell hybrids with the non-fused mixture of DC and tumor cells directly in their ability to induce a specific immune response. METHODS: Hybrids were obtained by electrofusion of tumor cells and monocyte-derived DCs. Cell phenotype was evaluated by flow cytometry and antigen-presenting ability by co-culture with syngeneic T cells followed by tetramer analysis and interferon (IFN)-γ ELISPOT. RESULTS: Less than half the cells in the mixture expressed DC co-stimulatory molecules. Furthermore, DCs in the mixture had significantly lower expression of MHC class I molecules than DCs in the fusion. Conversely, nearly all CD11c(+)Her2/neu(+) hybrids expressed CD80, CD86, CD83, HLA-DR and MHC class I from both tumor cells and DCs. Using tumor cells constitutively expressing a cytomegalovirus (CMV) antigen, we show that expansion of CMV-specific cytotoxic T lymphocytes (CTLs) restricted by DCs' MHC class I molecules was higher when DC-tumor hybrids were the stimulators. Furthermore, only hybrids stimulated CTLs to produce IFN-γ in response to CMV-positive target cells. CONCLUSIONS: These data show the superiority of DC-tumor cell hybrids over their simple mixture as T-cell stimulators. Hybrids expressed more co-stimulatory and MHC molecules, induced higher antigen-specific T-cell expansion and were the only cells able to induce IFN-γ-producing antigen-specific T cells. Thus, these data offer further support for cancer immunotherapeutic approaches using DC-tumor cell hybrids.

Immune response to cancer and its regulation in regional lymph nodes.

Regional draining lymph nodes (LNs) play a pivotal role in initiating immune responses. However, the presence of metastases may compromise their normal immunological function. Preclinical studies indicate that despite metastases, early tumor-draining LNs are still a rich source of sensitized T cells. Recently, we found that dendritic (DC)-tumor fusion hybrids were capable of stimulating therapeutic T-cell generation in the LN. However, this response is regulated by a tumor-specific suppression mechanism(s). Reversal of these dysfunctions would help the success of immunotherapy.

Megadose of T cell-depleted bone marrow overcomes MHC barriers in sublethally irradiated mice.

Graft-versus-host disease (GVHD) is uniformly lethal in recipients of HLA-mismatched marrow. In patients with severe combined immunodeficiency disease, this major obstacle can be overcome by rigorous T-cell depletion before transplantation. In leukaemia patients, however, the benefit of preventing GVHD is offset by graft rejection or graft failure. Very recently, this problem was overcome by supplementing T cell-depleted bone marrow transplants with megadoses of peripheral blood stem cells collected by leukapheresis after mobilization of the donor stem cells with granulocyte colony-stimulating factor (G-CSF). In the present study, we further demonstrate in a mouse model (C57BL/6-->C3H/Hej) that escalation of bone marrow doses by four- to fivefold leads to full donor-type chimerism in sublethally irradiated (6.5 Gy) recipients. Thus, the new source of G-CSF mobilized human haematopoietic stem cells may enable extending the use of mismatched bone marrow transplants to patients with non-malignant diseases for whom supralethal conditioning is not a prerequisite.

Regression of human metastatic renal cell carcinoma after vaccination with tumor cell-dendritic cell hybrids.

Reports of spontaneous regressions of metastases and the demonstration of tumor-reactive cytotoxic T lymphocytes indicate the importance of the host's immune system in controlling the devastating course of metastatic renal cell carcinoma. Recent research indicates that immunization with hybrids of tumor and antigen presenting cells results in protective immunity and rejection of established tumors in various rodent models. Here, we present a hybrid cell vaccination study of 17 patients. Using electrofusion techniques, we generated hybrids of autologous tumor and allogeneic dendritic cells that presented antigens expressed by the tumor in concert with the co-stimulating capabilities of dendritic cells. After vaccination, and with a mean follow-up time of 13 months, four patients completely rejected all metastatic tumor lesions, one presented a 'mixed response', and two had a tumor mass reduction of greater 50%. We also demonstrate induction of HLA-A2-restricted cytotoxic T cells reactive with the Muc1 tumor-associated antigen and recruitment of CD8+ lymphocytes into tumor challenge sites. Our data indicate that hybrid cell vaccination is a safe and effective therapy for renal cell carcinoma and may provide a broadly applicable strategy for other malignancies with unknown antigens.

Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl. XI. Pseudogenetic restrictions of hybridoma suppressor factors.

Suppressor factor derived from three different murine T cell hybridomas were characterized . They specifically inhibited 4-hydroxy-3-nitrophenyl acetyl cutaneous sensitivity responses. The factors bind antigen and bear I-J and idiotypic determinants, but lack conventional immunoglobulin constant-region determinants. The factors function during the induction phase of the immune response, by inducing a second population of suppressor cells (Ts(e)). Suppressor factor can inhibit both cellular and plaque-forming cell responses in appropriate strains of mice. These hybridoma suppressor factors directly suppress strains of mice that are Igh-V homologous with the strain producing the factor. Thus, there is an apparent Igh-V restriction in the activity of these factors. However, this is a pseudogenetic restriction because these factors generate second order suppressor cells (Ts(e)) in Igh-incompatible mice, but in order to express the suppressive activity, the cells must be adoptively transferred into recipients that are Igh compatible with the strain producing the suppressor factor. Finally, it was shown that the factor-induced Ts(e) population is under an apparent dual genetic restriction. Thus, Igh and H-2 homology is required in order for the Ts(e) population to express its suppressive activity.

Target-effector interaction in the human and murine natural killer system: specificity and xenogeneic reactivity of the solubilized natural killer-target structure complex and its loss in a somatic cell hybrid.

Preincubation of natural killer (NK) cells with electrophoresis purified proteins from a variety of NK-sensitive murine and human tumor cells specifically prevented subsequent binding to the intact, homologous target cell. The NK-target structures (NK-TS) consisted of some or all of four characteristic molecular species, tentatively assigned molecular weights of 140K, 160K, 190K, and 240K (+/-10K) based on electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels. When these NK-TS molecules were compared in cross-inhibition assays, the large 240K molecule most often carried the unique NK specificity, whereas the smaller 140K molecules cross-reacted between YAC, 136-6 and X-63 in the mouse and between Molt-4 and K562 in the human. Mouse NK cells recognised a different spectrum of NK-TS molecules than human NK cells. The control of NK-TS expression was partially revealed in a cloned, somatic cell hybrid bwtween an NK sensitive (YAC-IR) and insensitive (A9HT) cell line. The hybrid did not express NK-TS and did not bind to NK cells which is in accordance with negative NK cytolytic results previously reported. Although unique specificities are carried by some of the multiple NK-TS protein molecules, cross-reactions were widespread. These observations taken together suggest that the NK cell is polyspecific and has some heterogeneity in the recognition structure although much less than would be expected of an antibody-combining site.

Chromosome assignment of the tumor-specific antigen of a 3-methylcholanthrene-induced mouse sarcoma.

Chemically induced sarcomas of inbred mice express antigens that are distinct and specific for each individual tumor. Chromosome assignment of tumor-specific antigens would help to elucidate the genetic basis of their diversity. Expression of the serologically defined Meth A sarcoma antigen is not suppressed in hybrid cells containing a complete foreign (Chinese hamster) genome. Chromosome and serologic analysis of microcell hybrids between Meth A sarcoma cells and Chinese hamster cells shows a clear correlation of Meth A antigen expression with the presence of the distal region of chromosome 12 (bands F1 and F2). Chromosome 16 may also be implicated. The significance of the close linkage of genes determining Meth A antigen expression with the immunoglobulin heavy chain gene cluster (on chromosome 12) and the lambda light chain genes (on chromosome 16) is discussed.

Antigen-reactive T cell clones. II. Unique homozygous and (high responder x low responder)F1 hybrid antigen-presenting determinants detected using poly(Tyr, Glu)-poly D, L-Ala--poly Lys-reactive T cell clones.

Using murine (T,G)-A--L-reactive T cell clones, we have demonstrated the existence of unique homozygous antigen-presenting determinants expressed on C57bl/6 mice, controlled by the I-A subregion of the murine major histocompatibility complex (MHC), which are not expressed on semisyngeneic (C57Bl/6 x A/J)F1 [(B6A)F1] cells. Additionally, we were able to demonstrate that there exist (T,G)-A--L-reactive clones in F1 mice derived between low responder and high responder parents [(B6A)F1] that recognize antigen in association with transcomplementing hybrid I-A subregion determinants expressed uniquely on (B6A)F1 cells not expressed on cells of either of the parental strains. These data suggest that phenotypic high responsiveness exhibited by (higher responder x low responder)F1 mice was not simply controlled by the high responder parental genome, but was controlled at the phenotypic level of expression of antigen-presenting determinants. Such antigen-presenting determinants can be created by complementation using products of the low responder as well as high responder genome. The significance of the existence of such F1 specific hybrid antigen-presenting determinants for T cell specificity and recognition of self was discussed.

Maternally transmitted antigens are codominantly expressed by mouse cells containing two kinds of mitochondrial DNA.

Mtf, a cytoplasmic, probably mitochondrial factor, controls Mta polymorphism. We tested for dominance between two forms of Mtf to determine whether Mta is controlled by positive or negative genetic mechanisms. We fused Mtf-disparate cells containing distinct mtDNA markers and selected for hybrids containing both. Such mtDNA heteroplasmons codominantly and stably express alternative Mta antigens. Stable codominance excludes negative genetic mechanisms as well as a model of induced nuclear compensation, and implies Mtf controls Mta expression through a positive genetic mechanism.

Generation of anti-type III pneumococcal polysaccharide hybridomas from mice with an X-linked B-lymphocyte defect.

(CBA/N X BALB/c male)F1 mice bear on X-linked defect making them totally unresponsive to T-independent (TI), TI-2 antigens such as type III pneumococcal polysaccharide (SSS-III). We found that somatic cell hybrids between CB nonresponder spleen cells and NS1 plasmacytoma cells secreted antibody specific for SSS-III. The solid-phase binding of such antibody was completely inhibited by the addition of free antigen (SSS-III) and the amount of antibody detected in culture fluids ranged from 10 ng/ml to 10 micrograms/ml. Eight hybridoma clones were identified; all make antibody of the IgM class. These results indicate that the X-linked defect does not result in a deletion of a B-cell subset which responds to TI-2 antigens.

Anti-immunoglobulin antibodies. I. Expression of cross-reactive idiotypes and Ir gene control of the response to IgG2a of the b allotype.

The anti-allotype antibody response to the b allotypic form of IgG2a is regulated by major histocompatibility complex (MHC)-encoded immune response (Ir) genes. Mice of d, b, p, q, r, and s haplotypes make a strong anti-allotype response on immunization with the CBPC101 myeloma protein (IgG2ab), whereas mice of the k, m, a, a1, u, and z haplotypes made no, or a very poor, response. All responder strains produce anti-IgG2ab antibodies which share common idiotypes (Id) without relation to the allelic forms of the Ig heavy-chain-constant region genes that the responding mice possess. Isoelectric focusing analysis of the anti-allotype antibodies produced in various strains of mice showed that they are of limited heterogeneity and quite similar from strain to strain. Five out of six hybridoma products with specificity for CBPC101 allotype expressed cross-reactive idiotypes (IdX). Two of hybridoma products expressing IdX identify CH3-domain determinants, and one has been assigned a CH2-domain specificity.

Monoclonal rat anti-major histocompatibility complex antibodies display specificity for rat, mouse, and human target cells.

24 monoclonal rat antibodies are described that are reactive with determinants encoded by the major histocompatibility complex (MHC) of the rat. These hybridoma antibodies were derived by fusing mutant mouse myeloma cells to spleen cells from Lewis rats immunized with allogeneic Brown Norway cells. All 24 antibodies are cytotoxic for both Brown Norway target cells and target cells from the appropriate MHC congenic rats. Pattern of cytotoxicity and hemagglutination strongly suggest reactivity against class I (K or D equivalent) rat MHC determinants. Cytotoxic cross-reactivity patterns were generated for each monoclonal antibody on a panel of rat and mouse lymphoid cells and human peripheral T lymphocytes. A high degree of interspecies cross-reactivity was noted with approximately one-half of the antibodies positive on human and/or mouse target cells. 11 antibodies recognized polymorphic determinants in the mouse, and, by using target cells from MHC congenic mouse strains, it was shown that these determinants are encoded by genes within the H-2 complex. Finally, by considering the overall reactivity patterns of these monclonal antibodies on all target cells, one can show that these 24 antibodies represent a minimum of 14 antibody specificities.

Clonal analysis of F1 hybrid helper T cells restricted to parental or F1 hybrid major histocompatibility determinants.

Antigen-specific major histocompatibility complex (MHC)-restricted helper T cell precursors were induced to proliferate in cultures of keyhole limpet hemocyanin-primed lymph node cells. Clones of F1 hybrid helper T cells were isolated in limiting-dilution cultures. Each positive culture at a limiting-dilution of lymph node cells gave rise to > 10 helper T cells with a single MHC-restricted specificity. This made it possible to independently assay specific helper activity of isolated clones in secondary cultures with B cells of diverse origin. Different clones with helper activity restricted to either parental or unique F1 hybrid MHC determinants were found to occur at approximately equal frequency. The results are discussed in relation to hybrid Ia specificities and dual-complementing MHC-linked Ir genes.

T lymphocyte-mediated suppression of myeloma function in vitro. III. Regulation of antibody production in hybrid myeloma cells by T lymphocytes.

To investigate the mechanisms by which T lymphocytes regulate myeloma function in vitro, the effects of regulatory T cells on antibody secretion by a hybrid myeloma cell line were examined. Suppressor T cells (Ts) specific for idiotypic determinants on M315 (IgA, lambda 2 anti-2,4-dinitrophenol and anti-2,4,6-trinitrophenol [TNP]) and MPC 11 (IgG2b, kappa) myeloma proteins inhibit antibody secretion by the appropriate parental myeloma cells. When cocultured with a hybrid cell line derived by fusion of MOPC 315 and MPC 11 myelomas, the idiotype-reactive Ts inhibit secretion of only the immunoglobulin (Ig) bearing the relevant idiotype. In contrast, syngeneic TNP-reactive cytolytic T lymphocytes (CTL) inhibit antibody secretion by TNP-binding MOPC 315 cells but not by MPC 11 cells in the presence of soluble TNP-keyhole limpet hemocyanin (KLH), and this inhibition probably represents a prelytic effect of the CTL. Such TNP-reactive CTL, in the presence of TNP-KLH, inhibit both IgA and IgG secretion by the MOPC 315-MPC 11 hybrid, which is consistent with a prelytic effect. Thus, myeloma hybrids are a useful tool for investigating the effector function of regulatory T cells. These results are discussed with reference to the mechanisms of action of regulatory T cells and their relevance to modulation of physiologic humoral immune responses.

T cell hybrids with arsonate specificity. I. Initial characterization of antigen-specific T cell products that bear a cross-reactive idiotype and determinants encoded by the murine major histocompatibility complex.

T cell hybrids have been constructed between the BW5147 thymoma cell line and A/J splenocytes from mice suppressed with the p-azophenylarsonte hapten. Three independently derived cloned lines have been chracterized. Each secretes or sheds a 62,000-dalton antigen-specific product bound by rabbit antisera directed against the arsonate cross-reactive idiotype. In addition, each of the antien-specific molecules contains determinants encoded within the I region of the murine major histocompatibility complex. Peptide mapping analysis indictes that, whereas these molecules are remarkably similar, each is individually distinct in primary structure. The availability of cloned T cell lines that produce antigen-specific idiotype-positive I region-containing products should facilitate a more thorough dissection of the interrelationships of T-B interctions in the arsonate idiotypic system.

Structural correlates of cross-reactive and individual idiotypic determinants on murine antibodies to alpha-(1 leads to 3) dextran.

For the first time V-region amino acid sequence differences have been correlated with the expression of cross-reactive and individual idiotypes through an analysis of 12 dextran-binding proteins. This correlation has been possible because of the apparent sequence identity of the corresponding lambda chains. Expression of a cross-reactive idiotype was localized to two residues and/or a carbohydrate in the second hypervariable region of the heavy chain. Two individual idiotypes correlate with the two amino acids within the third hypervariable region that comprises the D segment of the dextran-binding proteins. These results demonstrate that idiotype reagents can recognize two amino acid differences within V and D segments of classical variable regions. In anti-dextran antibodies, cross-reactive idiotypes involve V-region determinants, whereas individual idiotype determinants correlate with D-segment variation.

Major histocompatibility complex-restricted self recognition. A monoclonal anti-I-Ak reagent blocks helper T cell recognition of self major histocompatibility complex determinants.

The functional role of cell surface Ia antigens has been studied for in vitro antibody responses, using as a probe the ability of anti-Ia reagents to inhibit these responses. A hybridoma monoclonal anti-Ia reagent specific for a product of I-Ak (Ia.17) profoundly inhibited in vitro antibody responses to TNP-KLH by spleen cells of the I-Ak but not I-Ab haplotype. This inhibition by anti-I-Ak product, but not by interaction with T or B cell product, in spite of the fact that functional B cells as well as accessory cells could be shown to express the determinant detected by this hybridoma reagent. These results suggest that the Ia expressed by accessory cells in of unique functional importance in these responses. To further characterize the function of Ia antigens in this response system, the mechanism of anti-I-Ak inhibition was determined. The inhibition resulting from interaction of anti-I-Ak with accessory cell Ia was not mediated by nonspecific suppressor cells, nor was there nonspecific interference with accessory cell function as a result of the binding of anti-Ia antibody. The relationship between anti-Ia inhibition and T helper cell recognition of self determinations on accessory cells was analyzed using T cells from radiation bone marrow chimeras. It was demonstrated that (B10 X B10.A)F1 leads to B10 (F1 leads to B10) chimera T cells were able to cooperate with B10 (H-2b and I-Ab) but not B10.A (H-2a and I-Ak) accessory cells for responses to TNP-KLH; F1 leads to B10.A T cells were able to cooperate with B10.A but not B10 accessory cells; and both chimera populations were able to cooperate with (B10 X B10.A)F1 (F1) accessory cells. Monoclonal anti-I-Ak inhibited the cooperation of F1 leads to B10.A T cells with the same F1 accessory cells. Thus, inhibition by anti-I-Ak is dependent upon active helper T cell recognition of I-Ak-encoded determinants expressed on accessory cells. These findings demonstrate that T cells recognize self Ia determinants expressed on accessory cells, and that such recognition is required for the generation of T cell-dependent antibody responses.

Monovalent fragments (Fab) of monoclonal antibodies to a sporozoite surface antigen (Pb44) protect mice against malarial infection.

Monoclonal antibodies (IG1, k) directed against a surface component of Plasmodium berghei sporozoites (Pb-44) confer complete protection to mice against a lethal inoculum of parasites. The degree of protection is a function of the number of parasites used in the challenge and of the antibody concentration in serum. Passive transfer of 10 micrograms of antibody per mouse abolished or profoundly diminished the infectivity of 10(3) sporozoites, but much higher amounts of antibody were required for complete protection against challenge with 10(4) parasites. Fab fragments of the monoclonal antibodies were as effective as the intact antibodies in mediating protection as determined by the neutralizing assay. This observation suggests that the antibodies interfere with a parasite function necessary for its infectivity, such as, for example, the ability to penetrate into the target cell or to multiply in the hepatocytes. When sporozoites are incubated with the intact monoclonal antibodies at 37 degrees C, a long filament appears at its posterior end (circumsporzoite precipitation [CSP] reaction). Fab fragments are ineffective at high concentrations. However, if after treatment with Fab, the sporozoites are incubated with rabbit antibodies to mouse k-chains, a strong CSP reaction is observed. We conclude that the CSP reaction can result from the cross-linking of Pb44 and that it has the characteristics of a capping reaction followed by the shedding of the immune complexes.

Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors.

To investigate the antigenic relationship between the macrophage and lymphocyte Fc receptors (FcR), a monoclonal antibody capable of blocking mouse macrophage Fc receptors was selected. Hybrids were formed by fusing the P3U1 mouse myeloma and spleen cells from a rat immunized with the mouse macrophage-like cell lines J774 and P388D1. The Fab fragment of the monoclonal IgG secreted by clone 2.4G2, inhibited the trypsin-resistant Fc receptor II (FcRII), which is specific for immune aggregates of mouse IgG1 and IgG2b, but had no inhibitory effect on the trypsin-sensitive Fc receptor I (FcRI), which binds monomeric IgG2a and erythrocytes coated with IgG2a. Thus, the monoclonal 2.4G2 IgG appeared to be specific for macrophage FcRII. Further evidence that the 2.4G2 IgG was directed against FcRII came from binding studies of the monoclonal antibody to J774 cells and a series of independently isolated variants which do not express FcRII. These variants of J774 bound 5% as much of the monoclonal antibody as the parent line, which bound 600,000 molecules of 2.4G2 IgG per cell. The antigenic relatedness of mouse lymphocyte FcR to mouse macrophage FcRII was demonstrated by the binding of 2.4G2 IgG to FcR-bearing lymphoid cell lines and the inhibition of the lymphocyte FcR by the monoclonal antibody. Preincubation of spleen cells and peritioneal cells with 2.3G2 IgG likewise inhibited rosette formation with ox erythrocytes coated with rabbit IgG. The ability of the hybridoma IgG to inhibit mouse FcRII was independent of the major histocompatibility complex. The 2.4G2 IgG antigenic determinant was not present on rat, guinea pig, rabbit, or human FcR-bearing cells.