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Two siblings with a peptide TAP deficiency were recently described. Despite poor cell surface expression of HLA class I molecules, these patients were not unusually susceptible to viral infections. The majority of the cell surface-expressed class I molecules were HLA-B products as assessed by cytofluorometry and biochemical analysis. Analysis of two peptides eluted from the class I molecules expressed by TAP-deficient EBV B lymphoblastoid cell lines indicated that both were derived from cytosolic proteins and presented by HLA-B molecules. Peripheral alphabeta CD8+ T cells were present and their TCR repertoire was polyclonal. Most of the alphabeta CD8+ T cell clones studied (21 of 22) were nonreactive against cells expressing normal levels of the same HLA alleles as those of the TAP-deficient patients. However, it was possible to isolate one cytotoxic CD8+ alphabeta T cell clone recognizing the EBV protein LMP2 presented by HLA-B molecules on TAP-deficient cells. These observations suggest that in the TAP-deficient patients, CD8+ alphabeta T cells could mature and be recruited in immune responses to mediate HLA class I-restricted cytotoxic defense against viral infections. They also strengthen the physiologic importance of a TAP-independent processing pathway of the LMP2 protein, which was previously shown to contain several other TAP-independent epitopes.

We previously demonstrated the occurrence of a naturally arisen human anti-idiotypic B cell clone, that we transformed with EBV (EBV383). We show evidence that EBV383 not only expresses the CD5 surface Ag, but also contains the 2.7-kb mRNA transcript encoding this protein. In addition, we show the presence of the 3.6-kb mRNA precursor. Most Ig produced by CD5+ B cells are polyreactive natural IgM antibodies encoded by unmutated copies of germline VH genes. However, in this study we present data demonstrating the monoreactive high affinity character of the anti-idiotypic antibody (mAb383) produced by EBV383. These data are in agreement with our previous observations, showing that the VH chain of mAb383 is encoded by an extensive somatically mutated VHV gene in a way that is consistent with an Ag-driven immune response. A possible role for this remarkable anti-idiotypic antibody in the maintenance of B cell memory is discussed.

We previously described the isolation and characterization of a human monoclonal anti-idiotypic antibody (Ab2) isolated from EBV-transformed human PBL after immunization with rabies vaccine. The present study concerns the molecular characteristics of the Ab2 and the germ-line elements that gave rise to it. The H chain of this antibody derives from the small VHV family of human V region gene segments. Parallel studies on the germ-line VHV gene isolated from the same individual revealed that the expressed molecule contains 19 nucleotide differences in the VH gene segment. The D segment of Ab2 could have arisen by a D to D fusion; the J segment is a JH6. Extensive somatic variation evident in the H chain variable region of this naturally arising monoclonal anti-idiotypic antibody suggests that this Ab2, the product of a CD5+ B cells, was the consequence of an Ag-driven immune response.

Stimulation of PBMC, in children recovering from acute measles, with autologous EBV-transformed and measles virus (MV)-infected lymphoblastoid cell lines (B-LCL) expanded primarily MV-specific CD8+ T cells. A large number of CD8+ T cell clones were obtained either by passaging of bulk cultures at limiting dilutions or by direct cloning of PBMC without previous stimulation in bulk culture. The MV-specific CD8+ T cell clones responding in a proliferative and a CTL assay were found to be class I MHC restricted. In contrast, CD4+ MV-specific T cell clones, which were generated by the same protocol, recognized MV in association with class II MHC molecules. Analysis of processing requirements for Ag presentation to CD8+ and CD4+ T cell clones, measured by the effect of chloroquine in a proliferative T cell response, revealed that both types of T cells recognized MV Ag processed via the endogenous/cytoplasmic pathway. Thus, these studies indicate that, as in most other viral infections and in contrast to previous suggestions, the class I MHC-restricted CTL response by CD8+ T cells may be an important factor in the control and elimination of MV infection. Therefore, the role proposed for CD4+ class II-restricted T cells in recovery from measles needs to be reevaluated.

Five asymptomatic human immunodeficiency virus (HIV)-seropositive male homosexuals were immunized with the recall antigens tetanus toxoid (TT) and the three types of poliovirus present in diphtheria, tetanus, and polio vaccine. Four weeks after immunization, the in vivo response to booster immunization, the in vitro pokeweed mitogen (PWM)-induced IgG secretion, and the in vitro T cell-dependent and T cell-independent antigen-induced antibody response were assayed. Increase in serum antibody titer to TT and poliovirus was low and normal, respectively. In all five subjects studied, a high rate of spontaneous IgG production, including antibodies directed toward HIV was observed. Addition of PWM to the cultures induced suppression of the spontaneous IgG secretion. Only one donor showed a slightly increased IgG production after stimulation with PWM. Peripheral blood mononuclear cells of four of the five HIV-seropositive individuals did not produce TT, or poliovirus-specific antibodies when stimulated with the respective T cell-dependent antigens. However, stimulation of these peripheral blood mononuclear cells with TT coupled to agarose beads, which was shown to be T cell-independent, resulted in the generation of IgG anti-TT antibody-forming cells.

In the present report an in vitro method for obtaining a secondary human antibody response to a dog kidney cell vaccine against rabies virus (DKCV) is described. Cultures of peripheral blood mononuclear cells from normal rabies-immune and nonimmune donors were stimulated in vitro by DKCV. The production of virus-specific antibody in supernatant fluids was monitored by ELISA. Antibody was produced by lymphocytes from rabies-immune individuals, whereas those of nonimmune subjects consistently failed to produce anti-rabies antibodies after in vitro stimulation with DKCV. The generation of the anti-rabies virus antibody response of lymphocytes stimulated with DKCV was shown to be an antigen-dependent, as well as an antigen-specific process. Optimal antigen-specific responses were observed at relatively low concentrations of antigen (10(-1) to 10(-2) micrograms/culture). At increasing concentrations of antigen in culture (greater than 1 microgram/culture), the anti-rabies virus response was suppressed. Antibody produced upon stimulation was capable of neutralizing rabies virus. The response to rabies virus requires T cell help because lymphocytes depleted of SE rosetting cells did not respond to an antigenic stimulus. Studies in which the same individuals were followed over time showed a sequential development of circulating B cell subsets. The system may provide a model for the study of human B cell differentiation in vivo and in vitro and may be valuable for testing the potency of rabies vaccines in vitro.

The in vitro induction of an ovalbumin-specific human T cell suppressor factor is described (TsF120-OA). The antigen-specific suppressive component can be purified by affinity chromatography from supernatants derived from Marbrook-Diener type cultures of peripheral blood T cells stimulated with a high dose of ovalbumin. TsF120-OA suppresses the antigen-induced PFC formation of human blood B cells in vitro in an antigen-specific way. The target of TsF120-OA activity is shown to be the T helper cell. No genetic restriction in the action of the factor is observed.

We intended to investigate whether the suppression of antigen-induced antibody responses in vitro in man by T suppressor cells required contact of T suppressor cells with target cells or whether this effect was mediated by factors released by T suppressor cells. To this end supernatants of antigen-induced T suppressor cells were tested (by a plaque forming cell assay) for their capacity to suppress antibody responses of autologous and allogeneic human peripheral blood lymphocytes. We have shown that supernatants of antigen-specific T suppressor cells, designated as TsF24: a) can suppress an antibody response of autologous but not allogeneic lymphocytes to the inducing antigen; b) are antigen-specific in their effect; and 3) are produced by radiosensitive T cells. Furthermore, the target of the factor is a radiosensitive T cell. These findings taken together indicate that, in the generation of T-effector suppressor cells in man, T-T interactions occur, and in addition, that cellfree factors may be involved in these interactions.

An effective candidate subunit vaccine consisting of the gp 70/85 of feline leukemia virus (FeLV) was prepared by using the immunostimulating complex (iscom) method for the presentation of membrane proteins of enveloped viruses. Two 32-wk-old specific pathogen-free (SPF) cats were immunized with a FeLV iscom vaccine prepared from the supernatant fluid of the FL74 tumor cell line without adjuvant. Both cats developed FeLV serum antibodies, as measured in an enzyme-linked immunosorbent assay (ELISA) and in a virus neutralization test. A proportion of the antibodies were directed to an epitope located on gp70/85, which was shown in competition ELISA with a peroxidase-labeled virus-neutralizing monoclonal antibody to be shared by all three subtypes of FeLV. The protective effect of FeLV iscom was studied by vaccinating six 8-wk-old SPF cats with iscom prepared from cell culture supernatant of another tumor cell line F422, followed by oronasal challenge with 10(6) ffu FeLV-A (strain Glasgow-1). Six unvaccinated cats were also challenged with the same dose of FeLV. The vaccinated cats developed FeLV serum antibodies, some of which were directed to the shared epitope on gp70/85. At 10 wk after challenge, none was viremic, whereas three of the control cats had developed FeLV viremia. The potential of FeLV iscom as a vaccine against FeLV-associated disease in cats, and of iscom vaccines for protection against mammalian retrovirus infections, is discussed.

Syngeneic monoclonal anti-idiotope antibody Ab2,2-17C3SCC was raised against an idiotope on a protective monoclonal antibody with specificity for poliovirus type II. Ab2,2-17C3SCC detects a paratope-related interspecies IdX. Ab2,2-17C3SCC purified from supernatant fluids of hybridoma cells by protein A-Sepharose was injected into 4- to 6-wk-old BALB/c mice. The sera of the mice were screened for the expression of antibodies bearing the corresponding idiotope. Immunization of mice with Ab2,2-17C3SCC induced antibodies of complementary specificity. Furthermore, micro VN tests suggest that Ab2,2-17C3SCC can substitute for antigen in the induction of anti-polio neutralizing antibodies, and hence can function as a monoclonal anti-idiotypic vaccine.

An in vitro system of poliovirus-specific antibody production by peripheral blood B cells on stimulation by the virus has been developed. Virus-neutralizing antibodies in culture supernatant fluids, or virus-specific antibody-secreting cells (ASC) were detected by microneutralization assay and ELISA-SPOT test, respectively. After booster immunization with polio vaccine, anti-poliovirus-neutralizing ASC were present in circulation. This response was measurable between 5 and 12 days after booster vaccination. At between 12 and 90 days, another subset of B cells was found in peripheral blood that only produced poliovirus-specific neutralizing antibody after in vitro antigenic stimulation. The in vitro virus-induced response required B cells, monocytes, and T4+ (T helper) cells, and was shown to result from de novo protein synthesis. The anti-poliovirus-neutralizing response in vitro could be dissected in a type-specific and intertypic cross-reactive response by using various antigen concentrations for in vitro stimulation. Evidence was obtained by absorption studies for the existence of intertypic cross-reactive neutralization-inducing epitopes.

The in vitro induction and assay of an ovalbumin-specific human T cell helper factor are described. Peripheral blood T cells, cultured with ovalbumin in a Marbrook-Diener system, produce an antigen-specific factor(ThF120-OA), which can be purified by affinity chromatography. The in vitro studies with ThF120-OA pointed out that in the production of the factor as well as in the factor-B cell interaction the adherent cell determines the genetic restriction. The results of kinetic studies on T helper activities demonstrated that Thf120-OA provides an auxiliary activity at various moments during the differentiation of the human peripheral B cell into an antibody-secreting cell. The observed differences in the mode of action of Th cells and Th factor are discussed.

PBMC from healthy adult individuals seropositive for measles virus (MV) were tested for their capacity to proliferate to UV-inactivated MV (UV-MV) or to autologous MV-infected EBV-transformed B cell lines (EBV-BC). MV-specific T cell responses were observed in 11 of 15 donors tested (stimulation index greater than 2), when optimal doses of UV-MV were used in proliferative assays. T cell clones were generated from PBMC of three donors responding to MV, by using either UV-MV or MV-infected autologous EBV-BC as APC. Stimulation with UV-MV generated exclusively CD3+ CD4+ CD8- MV-specific T cells, whereas after stimulation of PBMC with MV-infected EBV-BC, both CD3+ CD4+ CD8- and CD3+ CD4- CD8+ MV-specific T cell clones were obtained. Of 19 CD4+ T cell clones tested so far, 7 clones reacted specifically with purified fusion protein and 1 with purified hemagglutinin protein. Seven clones proliferated in response to the internal proteins of MV. Three clones reacted to whole virus but not to one of the purified proteins, whereas one clone seemed to recognize more than one polypeptide. Some of the T cell clones, generated from in vitro stimulation of PBMC with UV-MV, failed to recognize MV Ag when MV-infected EBV-BC were used as APC instead of UV-MV and PBMC. CD3+ CD4+ CD8- T cell clones recognized MV in association with HLA class II Ag (HLA-DQ or -DR), and most of them displayed CTL activity to autologous MV-infected EBV-BC. All CD4+ HLA class II-restricted CTL clones thus far tested were capable of assisting B lymphocytes for the production of MV-specific antibody. The CD4- CD8+ T cell clone MARO 1 recognized MV in association with HLA class I molecules and displayed cytotoxic activity toward MV-infected EBV-BC.

Measles virus (MV)-specific murine helper T cell clones (Thy-1.2+, CD4+, CD8-) were generated from mice immunized with MV-infected mouse brain homogenate by limiting dilution and in vitro stimulation of spleen cells with UV-inactivated MV Ag. The protein specificity of 7 out of 37 stable T cell clones, which displayed MHC-restricted MV Ag recognition, could be assessed by using purified MV proteins. Two fusion (F) protein-specific, two hemagglutinin-specific, and three nucleoprotein- or matrix protein-specific clones were shown to be established. The F protein-specific T cell clones together with a panel of previously generated F protein-specific T cell clones were characterized for their fine specificity by using beta-galactosidase fusion products, which contained different parts of the F protein. It was shown that at least two epitopes on the major part of the F protein (amino acid 2-513) can be recognized by mouse T cells. Functional characterization of three T cell clones showed that they were able to assist MV-specific B cells and bystander B cells for antibody production. Furthermore, they were shown to produce the lymphokines IL-2 and IFN-gamma. It was also shown that these T cell clones induced a MV-specific delayed type hypersensitivity response. These observations suggest that all of the T cell clones characterized belong to the TH1 helper subset.