Priming of T helper cells by antigen-activated B cells. B cell-primed Lyt-1+ helper cells are restricted to cooperate with B cells expressing the IgvH phenotype of the priming B cells.

Activated B cells isolated shortly after primary immunization of BALB/c donor mice with sheep erythrocytes (SRBC), were transferred to normal syngeneic recipients or to low-dose cyclophosphamide-pretreated syngeneic recipients. In pretreated recipients, the transfer of activated B cells, but not of T cells or macrophages, resulted in an augmented production of indirect plaque-forming cells in the primary immune response to SRBC but not to horse erythrocytes. It was shown in double-transfer experiments that T helper cells (Lyt-1+) had been stimulated by the transfer of antigen-activated B cells. Criss-cross double-transfer experiments using the mouse strains CB20 and BAB14 (congenic to BALB/c at the loci coding for the immunoglobulin heavy chain) indicate that those T helper cells are primed after recognition of B cell products that are encoded for by genes linked to the loci coding for the variable region of the immunoglobulin heavy chain (IgVH). The thus-primed Ig-dependent T helper cells (THIg) are adaptively restricted to cooperate with B cells that display IgVH-linked gene products similar to those that originally stimulated the THIg. These findings suggest that in the course of an immune response to T cell-dependent antigens, help for the production of specific IgG can be provided by THIg that have been primed and/or clonally expanded after recognition of IgVH-linked gene products by (e.g., complementary) T cell receptors.

Immunological memory in mice. I. Physical separation and partial characterization of memory cells for different immunoglobulin classes from each other and from antibody-producing cells.

Plaque forming cells (PFC) of different immunoglobulin classes producing antibodies against sheep erythrocytes were separated according to their buoyant densities by means of equilibrium centrifugation in a stepwise BSA gradient. In the period of 7-10 days after immunization gammaM PFC are markedly enriched in fractions of low density and relatively depleted in fractions of high density. The distribution of total gammaG PFC shows less enrichment in the lower density fractions and less depletion in the higher density fractions. The density profile for gammaG(2a) PFC is even flatter, with a significant difference (depletion) relative to the unseparated spleen cells only in the highest density fraction. The density gradient distributions of cells able to transfer an adoptive immune response of the various immunoglobulin classes are markedly different from the PFC distribution. Cells obtained 7-10 days after immunization able to transfer an IgM response are present in the same proportions across the density gradient, whereas memory cells for gammaG(2a) obtained at this time are markedly enriched in fractions of low density and virtually depleted from high density fractions. With increasing time after primary immunization, the gammaG(2a) memory cells increase progressively in density and by 6 weeks the higher and lower density fractions have the same proportions of gammaG(2a) memory cells. The total gammaG (mainly gammaG(1)) memory cells by 7-10 days show slight enrichment in low density fractions and no depletion in high density fractions. The conclusions were reached that (a) memory for gammaG(1) develops earlier than memory for gammaG(2a) and (b) that memory for anti-SRBC antibodies of different classes is carried in separate cells. When gradient fractions enriched for PFC and memory cells for all classes were completely depleted of PFC using glass bead columns, the ability of this fraction to transfer memory for all classes was not diminished. This shows that memory cells are not identical with cells secreting antibodies.

Immunological memory in mice. II. Cell interactions in the secondary immune response studies by means of immunoglobulin allotype markers.

Congenic mice, differing genetically only at the loci coding for immunoglobulin H chain (or Fc) structures, have been used to study cell interactions in the 7S (gammaG(2a)) antibody response to sheep erythrocytes (SRBC), as detected by the Jerne plaque-forming cell (PFC) method. The interaction between thymus and bone marrow cells was studied in adult thymectomized irradiated recipients, protected with syngeneic bone marrow and injected with thymus cells from the partner congenic strain. All of the gammaG(2a) PFC detected in the spleens of these mice were of bone marrow allotype. Adoptive secondary immune responses were then studied to determine whether a similar interaction between memory cells and bone marrow derived cells could be detected. Primed spleen cells from the partner congenic strain, or a subpopulation of these cells obtained by BSA density gradient fractionation, were injected into irradiated recipients alone, or together with syngeneic nonimmune spleen or bone marrow cells. All gammaG(2a) PFC detected in these experiments were of primed cell allotype. There was no evidence that antibody forming cell precursors in normal spleen or bone marrow participate in the adoptive secondary immune response detected 7 days after transfer of primed spleen cells. This was true regardless of whether the bone marrow cells were injected at the time of transfer, or were injected 1-2 wk earlier and allowed to become established in the spleens of recipient mice. Although no specific cell interaction was seen, bone marrow (and, to a lesser degree, normal spleen) cells were found to have a nonspecific enhancing effect on the adoptive secondary response when they were injected together with the primed spleen cells. This enhancement was not evident if the bone marrow cells were injected 1 or 2 wk prior to primed cell transfer.

Laboratory scale production of monoclonal antibodies in a tumbling chamber.

A simple device for laboratory scale production of monoclonal antibodies has been developed. Hybridomas were cultured in four individual dialysis tubes containing 40-50 ml medium with 10% foetal calf serum, surrounded by 1.5-2 litres supply medium without any serum supplement. Once placed on a roller the special design of the apparatus leads to an eccentric rotation, thus keeping the cells in a stable homogeneous suspension. The system is automatically gassed, and this makes long term cultivation possible. Several hybridomas were tested over a culture period of at least 3 weeks, with supply medium changes every 3-4 days. Cell densities of up to 2.5 x 10(7)/ml and antibody concentrations of 0.3-1.9 mg/ml after purification were obtained. Results with this in vitro system allow a complete renunciation of the established in vivo method. The so called 'tumbling chamber' apparatus is easy to handle and to sterilize, is economic and universally adaptable in any research laboratory.

Human immunodeficiency virus transmission by organ donation. Outcome in cornea and kidney recipients.

The human immunodeficiency virus (HIV) is reportedly transmitted by sexual contact, sharing of infected needles among intravenous drug abusers, blood and blood products, artificial insemination, and kidney transplantation. This study reports on cornea and kidney recipients of two HIV-infected donors. HIV was transmitted to two kidney recipients who developed symptoms of acute HIV infection (i.e., fever, leukopenia, mild thrombopenia, splenomegaly) starting 12 days after transplantation. These signs of acute infection ended with seroconversion of HIV antibodies on approximately the 56th day after transplantation. The three cornea recipients showed no signs of acute infection and no HIV antibodies were detected up to three years after transplantation. The nontransmission observed in our cases, however, may not be representative of cornea transplantations in general. HIV is neurotropic in the later stages of the disease, and transmission of other neurotropic viruses like rabies and Creutzfeldt-Jakob disease by cornea transplantation has been reported. All tissue and organ donors should be tested for anti-HIV prior to donation.

The effect of cyclosporine on the progression of human immunodeficiency virus type 1 infection transmitted by transplantation--data on four cases and review of the literature.

Two women and two men were infected with the human immunodeficiency virus type 1 (HIV-1) transmitted by renal transplantation from i.v. drug-addicted donors in 1984. The four recipients were treated with cyclosporine and methylprednisolone (one patient only for three months because of early graft failure). Two patients died 66 and 74 months after transplantation, one of endocarditis and one of cerebral hemorrhage. Despite several infections including urinary tract infection (n = 8), peritonitis (n = 1), shunt infection (n = 1), bronchitis (n = 1), salmonellosis (n = 1), herpes stomatitis (n = 2), herpes zoster (n = 1), and cytomegalovirus (n = 1), and despite treatment of several rejection episodes (n = 8), none of them had or has infections typical of the acquired immunodeficiency syndrome (AIDS). However, two patients developed cervical lymphadenopathy and one autoimmune thrombocytopenia 15-20 months after HIV-1 infection. Their T helper cell counts (355/microliters to 75/microliters) and helper/suppressor T cell ratios (1.0-0.2) are distinctly lowered. One patient has membranous glomerulopathy with virus-like particles within and on the outside of the basement membrane and tubuloreticular inclusions in glomerular endothelial cells. We evaluated the case reports of 53 patients with HIV-infection caused by an infected transplant or by blood transfusions during or shortly after transplantation. The cumulative incidence of AIDS was significantly lower in 40 transplant patients with an immunosuppressive regimen including cyclosporine than in 13 transplant patients receiving immunosuppressive treatment without cyclosporine (5-year cumulative risk of AIDS: 31% versus 90%, P = 0.001).

Comparative immunoelectron microscopy with monoclonal antibodies on yellow fever virus-infected cells: pre-embedding labelling versus immunocryoultramicrotomy.

To study the intra- and extracellular distribution of yellow fever virus 17D (YFV)-specific antigens, pre-embedding immunoelectron microscopy (IEM) and IEM on ultrathin frozen sections were carried out comparatively using monoclonal antibodies (MAB) and YFV-infected cells. In addition, three electron-dense marker systems (IgG-ferritin and IgG-gold and protein A-gold) were compared for their efficiency in detecting bound MAB. Pre-embedding immuno-labelling was performed in microtest plates followed by in situ embedding and immunocryoultramicrotomy was performed using pellets of sucrose-infused cells. In both procedures, cells were prefixed with different concentrations of glutaraldehyde (GA). In pre-embedding IEM virus-specific antigens could be detected on the envelopes of extracellular virions with YFV-neutralizing MAB. Using immunocryoultramicrotomy, neutralizing MAB bound to intracellular mature virions as well as to viral antigens incorporated into cytoplasmic membranes. A concentration of 1% GA destroyed antigenicity entirely, while with 0.25% and 0.1% GA immunoreactivity was retained for more than 3 mth. Some highly reactive MAB labelled antigen significantly in pre-embedding IEM, when used at concentrations of 1 ng/ml. Immunocryoultramicrotomy was 10-100 times less sensitive. On cryosections colloidal gold was the marker of choice, due to the fact that it showed less nonspecific sticking to intracellular components and that it was easily detectable on highly contrasted cryosections. Owing to their higher sensitivity, IgG-ferritin conjugates were preferred in pre-embedding IEM.

Studies on induction and control of cell-mediated autoimmunity. I. Induction of "autoreactive" T lymphocytes in mice by cyclophosphamide.

Injection of a single dose of cyclophosphamide (CY) (125 mg/kg) or a combination of a small dose of CY (20 mg/kg) and 2.5 mg/kg lipopolysaccharide induces a transient appearance of autoreactive T lymphocytes (T-ARC) in the spleens of mice. The T-ARC activity reaches a peak 6 days after CY injection and could not be detected 8 days after this treatment. For testing T-ARC activity, spleen cells were injected into the footpads of syngeneic recipients, and the resulting lymph node enlargement at the draining site of cell inoculation and the content of nucleated cells in the lymph node was determined. Possible explanations of this autoimmune phenomenon are discussed. It is postulated that CY-resistant precursors of T-ARC are stimulated by "new" antigenic sites present on the surface of B lymphoblasts repopulating the CY-damaged spleen in a period of transient absence of CY-sensitive suppressor cells.

Studies on induction and control of cell-mediated autoimmunity. II. Prevention of induction and activity of autoreactive T cells by suppressor cells and by a suppressive serum factor.

Autoreactive T lymphocytes (T-ARC) can be detected in the spleen of mice treated with a single dose of cyclophosphamide (CY) (125 mg/kg), a peak occurring 6 days after CY injection. Eight days after CY treatment, the mice develop a specific anergic state. This anergic state can be transferred to normal syngeneic animals by either splenic nylon wool-nonadherent lymphocytes (suppressor cells, S-ARC) or by the serum of anergic mice, implying the development of an active suppressive mechanism due to CY treatment. Precursors of both potentially T-ARC as well as S-ARC coexist in the spleen of normal animals. Precursors of S-ARC present in the spleen and in the thymus of normal animals are sensitive to CY. However, committed S-ARC obtained from anergic mice are resistant to CY. Committed S-ARC as well as their precursors prevent induction of T-ARC. Committed S-ARC counteract expression of committed T-ARC activity, whereas precursors of S-ARC fail to do so. The autoimmune phenomenon described here represents an in vivo animal model system for induction of T-ARC and for the control mechanism which normally prevents induction and/or expression of cell-mediated autoreactivity by specific suppressor cells and by suppressive factors.

Suppression and potentiation of expression of delayed-type hypersensitivity by dextran sulphate.

Dextran sulphate delays the onset, or even completely suppresses the expression in mice of DTH or SRBC when administered via a route different from that of eliciting antigen. However, DS injected together with the eliciting antigen potential the expression of DTH. Dextran showed no effect on DTH. Cell transfer experiments suggest that the targets for the action of DS are the accessory cells (monocytes) and not the T-effector cells. As shown, using polystyrene latex particles and lipopolysaccharide from E. coli, trapping and perhaps activation of the trapped accessory cells rather than toxic effects of DS are responsible for these phenomena.

Antigen presentation, loss of immunological memory and AIDS.

A key factor causing immunodeficiency in HIV infection seems to be defective antigen presentation. Consequently, CD4+ T-cell populations, initially those expressing CD45RO, decrease in number not because of their destruction, but because they fail to expand in response to antigenic stimulation. This view implies that it would be mistaken to aim therapies only at correcting T-cell function or preventing infection of T cells.

Stimulation of regulatory T cell circuits by immunoglobulin-dependent structures on activated B cells.

An immunoregulatory circuit is described in which B cell blasts activate syngeneic Ly-1+2-3- T cells to (a) start a reaction which is indistinguishable from a graft-vs.-host reaction (syngeneic GvH) and (b) induce suppressor cell activity which abrogates the syngeneic GvH. Since capping the surface immunoglobulin (Ig) on B cell blasts blocks their ability to activate the circuit, it is likely that the relevant cell surface structure "seen" on the B cell by the Ly-1 T cell is either Ig itself or another molecule in association with Ig.

Development of viremia and humoral and cellular parameters of immune activation after vaccination with yellow fever virus strain 17D: a model of human flavivirus infection.

To monitor early and late events of immune system activation after primary and secondary flavivirus infection, 17 healthy persons were vaccinated with the standard 17D vaccine virus strain of yellow fever (YF). Twelve of these persons had not received YF vaccine previously and 5 had been vaccinated once at least 10 years before. Viremia and various parameters of humoral and cellular immune activation were followed daily for 7 days and weekly thereafter. Viremia was detected by reverse transcriptase-polymerase chain reaction in all 12 first-time vaccinees beginning from the second to the sixth day after vaccination; most tested positive between the fourth and sixth day. Infectious 17D virus was detected using a plaque forming assay in the serum of 7 of the 12 first-time vaccinees. As first parameters of immune activation, neopterin and beta2-microglobulin markedly increased between day 2 and day 6 postvaccination. In parallel to the viremia, circulating CD8+ T-cells significantly increased, with peak levels at day 5 after primary vaccination, indicating an activation of the cellular immune system. Neither viremia nor significant changes of these activation markers were observed in the five revaccinated persons. Neutralizing antibodies directed against the 17D vaccine strain developed in all persons within 2 weeks after vaccination. No correlation was found between the extent of viremia and the titer of neutralizing antibodies. Revaccination was followed by a minor and transient increase of neutralizing antibodies. High titers of neutralizing antibodies persisted for at least 10 years after primary vaccination.

Epitope mapping of monoclonal antibodies against human immunodeficiency virus type 1 structural proteins by using peptides.

Murine monoclonal antibodies directed against the structural proteins p17 and p24 of human immunodeficiency virus type 1 were investigated in an epitope mapping system. Overlapping peptides consisting of 15 amino acids of the p17 and p24 protein, respectively, were used as competitors in an enzyme-linked immunosorbent assay. Three different immunogenic regions (A, B, and C) could be defined, one on p17 and two on p24. Twenty monoclonal antibodies reacted with the human immunodeficiency virus type 1 peptides of region B, although differences in the reactivity of these antibodies with human immunodeficiency virus type 2 and simian immunodeficiency virus strain mac were detectable. Recognized epitopes were characterized by computer analysis as described by T.P. Hopp and K.R. Woods (Proc. Natl. Acad. Sci. USA 78:3824-3828, 1981) and P.Y. Chou and G.D. Fasman (Biochemistry 13:222-245, 1974).

CR1 (CD35) and CR3 (CD11b/CD18) mediate infection of human monocytes and monocytic cell lines with complement-opsonized HIV independently of CD4.

Peripheral blood and tissue mononuclear phagocytes serve as major viral reservoirs in HIV-infected individuals. We investigated the role of complement receptors CR1 (CD35) and CR3 (CD11b/CD18) in mediating productive infection with complement-opsonized HIV-1 and HIV-2 of cultured normal human peripheral blood monocytes, the promonocytic cell line THP-1, the monocytic cell line Mono Mac 6 and the glial cell line U251-MG. Cells were infected with the HTLV-IIIB strain of HIV-1 or the LAV-2 strain of HIV-2 that had been preopsonized with fresh human normal HIV seronegative serum. Productive infection was assessed by syncytia formation, the MTT cytotoxicity assay and/or release of p24 antigen in culture supernatants. Using suboptimal amounts of virus to infect the cells, we observed a higher and earlier productive infection of the cells with complement-opsonized HIV than with unopsonized virus. The enhancing effect of complement was totally suppressed by blocking CR1 or CR3 function with F(ab)'2 fragments of anti-receptor MoAbs; while blocking of the LFA-1 antigen had no effect. The infection of monocytic cells with complement-opsonized virus occurred independently of CD4 since it was not inhibited by F(ab)'2 fragments of a MoAb against the gp120 binding site of CD4 and since infection also occurred with Mono Mac 6 and U251-MG cells, which lack expression of the CD4 antigen and of CD4 mRNA. These observations suggest that complement may mediate productive infection of cells of the monocytic lineage with 'lymphocytotropic' HIV strains independently of CD4.

Infection of the human monocytic cell line Mono Mac6 with human immunodeficiency virus types 1 and 2 results in long-term production of virus variants with increased cytopathogenicity for CD4+ T cells.

The recently established human monocytic cell line Mono Mac6 expressing distinct characteristics of mature monocytes/macrophages was tested for its susceptibility to infection with human immunodeficiency virus. Inoculation of the cells with the T-cell-tropic human immunodeficiency virus strains human T-lymphotropic virus type IIIB and lymphadenopathy-associated virus type 2 led to a noncytopathic productive infection becoming apparent only after a latency period of up to 56 days. The infectibility of the Mono Mac6 cells was dependent on low levels of CD4 expression, as demonstrated by blocking experiments with various CD4-specific antibodies. Increasing with time after infection (greater than 200 days), the cultured Mono Mac6 cells released virus variants which showed shortened latency periods when passaged onto uninfected Mono Mac6 cells. Also, cytopathogenicity for several CD4+ T cells of the Mono Mac6-derived virus was drastically increased; thus, the infection of the H9 cell line with low doses of virus (less than 0.1 50% tissue culture infective dose per cell) led to giant syncytium formation within 1 day and subsequent death of all fused cells. We propose Mono Mac6 cells as a new model for the study of human immunodeficiency virus infecting the monocyte/macrophage lineage, particularly with regard to virus-host cell interaction and the influence of cell differentiation and activation on latency and development of virulence. The human immunodeficiency virus-infected Mono Mac6 cell may also serve as a valuable tool for in vitro testing of antiviral therapies.

Monoclonal antibodies directed against human immunodeficiency virus (HIV) gag proteins with specificity for conserved epitopes in HIV-1, HIV-2 and simian immunodeficiency virus.

Monoclonal antibodies (MAbs) were raised against gag proteins of human immunodeficiency virus type 1 (HIV-1), strain HTLV-IIIB. One of 29 antibodies was specific for p17 of HIV-1. Twenty of 28 MAbs reactive with the major core protein p24 of HIV-1 showed cross-reactivity with HIV-2, and five of these also detected the corresponding antigens of simian immunodeficiency virus (SIVmac). The MAbs were reactive in several tests, i.e. ELISA, immunostaining of Western blots, immunofluorescence, alkaline phosphatase-anti-alkaline phosphatase immunocytochemistry and immunoelectron microscopy. The submembrane protein p17 was clearly localized within the virion.