Electrophoretically homogeneous antibody synthesized by spleen foci of irradiated repopulated mice.

10(6) splenocytes from primed donors were injected, together with sheep erythrocytes (SRBC), into X-irradiated syngeneic mice. 8 days later the spleens were excised and cut into small fragments, keeping track of their location in the organ. Each fragment was cultured individually for 24 hr in the presence of (14)C amino acids and the culture fluids were assayed for antibody activity. The antibody-producing fragments were found to be clustered in few restricted areas (foci) surrounded by negative tissue. The anti-SRBC antibody from single foci was purified by absorption on stroma followed by acid elution. Thereafter, it was subjected to electrophoresis and immunoelectrophoresis. The radioautography of the runs showed a considerable degree of homogeneity. Distinct and sharp spikes were localized in the beta and gamma region. The pattern of each focus is unique from the point of view of the number of spikes and their mobility. Eluates obtained from many pooled fragments gave a broad radioactive smear in beta-gamma region. Many foci synthesized antibody migrating as a single band. This homogeneous protein is probably the product of a clone of cells homogeneously differentiated. However, some foci producing two and probably more antibody bands were also encountered. Two interpretations of the finding can be given. Either more than one precursor may participate in the formation of a focus or a differentiation switch may occur during the clonal expansion.

Induction of an antibody response in cultures of human peripheral blood lymphocytes.

A culture system is descirbed which provides adequate conditions for in vitro immunization of humand peripheral blood lymphocytes to heterologous erythrocytes. Making use of this method we could obtain, with a number of different donors, an antibody response which peaked at about day 8 of culture with 30-300 plaque-forming cells (PFC) per 10(6) input lymphocytes. However, in a number of experiments poor or negative results were obtained, even with donors that had previously given good response. This variability in the results was shown not to be due to a too low number of precursor cells present in the blood and could be overcome by treating the cells, before initiation of the culture, with a factor produced by mouse T cells educated to sheep erythrocytes (SRBC). Under these conditions a PFC responce was obtained which peaked at about day 8 and which in some experiments could be as high as 20,000 PFC per 10(6) input lymphocytes. Paralleling the increase in PFC was an increase in cell number. The cells recovered from the treated cultures were at all times more numerous than in the nontreated cultures. The height of both the proliferative and antibody-producing responses varied from experiment to experiment, a higher proliferative response, accompanying a higher PFC response. Although the mechanisms that are at the basis of the antibody response in vitro described in this paper still need to be clarified, this system may become a useful tool in studying the immune response in man.

Antibody response of rabbit blood lymphocytes in vitro. Kinetics, clone size, and clonotype analysis in response to streptococcal group polysaccharide antigens.

Peripheral blood lymphocytes (PBL) of rabbits previously hyperimmunized against streptococcal groups A and A-variant antigens were stimulated in vitro by the corresponding vaccines to produce group-specific antibody. This response was dependent on an optimal cell density (2 X 10(6) cells/ml), on the presence of antigen, it was specific and cross-reactive due to a shared rhamnose backbone of the two polysaccharide antigens, and it was highly selective, such that in a 42-55-day culture 1 out of 20 viable cells was a specific PFC. During the exponential increase of the antibody concentration at a constant number of PFC, antibodies were secreted at a rate of 2.4 X 10(4) molecules/s per cell until a plateau level of antibody (40 mug/culture) was reached. The microculture system was used to determine the minimal frequency of group polysaccharide-specific precursor cells in the blood. Independent of the time elapsed since the last immunization this frequency was 1-3 X 10(-5), i.e., in the range of 1-2.8 X 10(2) precursor cells per ml blood. This number was further used together with the clonotype analysis of the culture supernates to calculate the frequencies of precursors of major and minor clonotypes. A hierachy of persisting clonal memory precursor cells was found indicating that clonal dominance is determined by locked-in frequency patterns and therefore it is a phenomenon based on numbers of cells that respond to the antigen.

Primary antibody response of rabbit blood lymphocytes in vitro.

This paper describes a method for in vitro induction of a primary response of rabbit peripheral blood lymphocytes to sheep red blood cells. The response is measured by visualizing and enumerating the plaque-forming cells (PFC). Removal of an adhering suppressor cell and use of a low cell concentration in culture are among the crucial requirements. Maximum response was usually reached after 10--15 days of culture. The number of PFC then decreased or stayed at roughly plateau level at least up to the fourth week of culture, when most of the experiments were terminated. In several instances the response had a cyclical character with repeating peaks of PFC. Only plaques of the direct type were found.

A two-stage culture system for the induction of antibody-forming cell clones in cultures of normal human blood lymphocytes.

A two-stage culture method is described for the induction of a specific antibody response to sheep red cells (SRC) in microcultures at limiting dilutions of human peripheral blood lymphocytes (PBL). PBL from normal donors were cultured for 4 days with antigen and EBV using well defined conditions. The cells were then distributed in 10 microliter microcultures at different cell densities in order to estimate the frequency of responding units. The culture wells were tested for the presence of anti-SRC antibody by the spot test. The results show that the expression of antibody-forming cell clones in the second stage microcultures is strictly dependent on the presence of both antigen and EBV during the first stage cultures. The efficiency of the system was improved by the addition of 4% polyethylene glycol (PEG, MW 6000) in the first stage and its removal in the second stage and by the use of human serum (instead of fetal calf) in both stages. This approach permits the separation of different cellular events, occurring when human B cells are stimulated by antigen and represents a useful approach for studying the mechanisms of the specific immune response in man.

Induction of antibody forming cells with specificity for Candida albicans mannoproteins in cultures of human peripheral blood lymphocytes.

A method is described for the induction of a specific antibody response to Candida albicans in cultures of normal human peripheral blood lymphocytes (PBL). PBL were cultured in the presence of whole C. albicans cells and the antibody response was evaluated by the ELISPOT technique, on plastic wells coated with a purified candidal cell well mannoprotein (MP). Under the conditions described here, a specific antibody response was obtained in all of the eight donors tested. The response was antigen-dependent and antigen-specific, peaked around day 10-12 of culture and the antibodies belonged to both the IgM and the IgG isotypes. By testing the cultured cells on MP from different Candida species, the method permitted the detection of antibodies directed against MP epitopes shared by C. albicans and C. parapsilosis.

The antigen-specific induction of normal human lymphocytes in vitro is down-regulated by a conserved HIV p24 epitope.

Synthetic peptides containing amino acid sequence 218-238 of the core protein p24 of human immunodeficiency virus type 1 (HIV-1) and progressively shorter sequences at its C-terminus, were tested for their effect on antigen dependent in vitro responses of peripheral blood lymphocytes (PBL) from normal human donors. A peptide as short as 7 amino acids, corresponding to a highly conserved sequence, was able to inhibit in a dose-dependent manner the induction of a specific primary antibody response to the sheep red cell (SRC) antigen, as well as the proliferative response to recall microbial antigens. The results of this study constitute additional evidence of the immunoinhibitory effects of HIV components and may help to unravel some of the pathogenic mechanisms of AIDS. Moreover, they are of potential relevance for the development of immunoprophylactic and therapeutic strategies.

Induction of a specific antibody response to Bordetella pertussis antigens in cultures of human peripheral blood mononuclear cells.

The role of specific antibodies in protective immunity to Bordetella pertussis has not yet been clearly defined. In the present work, the induction of a specific antibody response to B. pertussis in cultures of human peripheral blood mononuclear cells (PBMC) was investigated, on the assumption that the capacity of circulating lymphocytes to mount a specific response in vitro may provide a useful parameter for the evaluation of protective immunity. When PBMC from normal adult donors were cultured with a heat-inactivated B. pertussis whole-cell suspension, cells secreting antibodies to pertussis toxin, pertactin and filamentous haemagglutinin were generated consistently. The antibody response peaked between days 7 and 11 of culture and the antibodies produced were exclusively of the IgM class.

A mannoprotein constituent of Candida albicans cooperates with antigen in the induction of a specific primary antibody response in cultures of human lymphocytes.

The effects of Candida albicans mannoproteins on the induction of a primary antibody response to a T-dependent antigen, sheep erythrocytes (SRBC), in cultures of human blood lymphocytes, were investigated. Two experimental systems (bulk and limiting dilution cultures) allowing the detection of both enhancing and inhibitory effects, were used. In bulk cultures, antigen alone elicited a small number of specific antibody forming cells, unless IL-2 was supplied. Addition of the fungal mannoprotein extract or of a purified constituent of it increased 5 to more than 10 times the specific response. When limiting dilution analysis was performed, we observed that: a) a similar number of specific precursor cells was induced by antigen and either IL-2 or mannoprotein; b) the plot of the number of seeded cells versus the log of the fraction of negative cultures was linear in antigen and IL-2 triggered cultures but constantly deviated from linearity when the candidal stimulant was added. Thus, more than one type of precursor cell was limiting in these cultures, and the immunoenhancing effect of mannoprotein may involve multiple cellular interactions.

Immunoregulatory effect of a synthetic peptide corresponding to a region of protein p24 of HIV.

The effect of a synthetic peptide, corresponding to a sequence of HIV-1 p24 protein (amino acids 218-237), on in vitro immune responses was studied. The peptide inhibited in a dose-dependent manner the induction of an anti-SRC antibody response and of a PPD-specific proliferative response of human PBL. On the other hand, PHA-induced proliferation of human PBL and PPD-induced proliferation of a PPD-specific human T-cell line were not modified by comparable amounts of the peptide. These results suggest that structures from a protein (p24), present in the serum throughout the course of HIV infection, are able to interfere with the inductive stages of specific immune responses. These findings may help to unravel some of the pathogenic mechanisms of AIDS and may contribute to the development of vaccine strategies.

Origin and development of a scientific collaboration and a long-lasting friendship.

The aim of this contribution is twofold: to honour Ivan Lefkovits with a short recollection of our scientific collaboration in the years 1972-1985 and a summary of our joint contribution to studies of the mechanisms and functions of the immune system and to acknowledge our long-lasting friendship. Ivan's limiting dilution microculture method was adapted to rabbit peripheral blood lymphocytes (PBL). The antibody produced in the responding cultures was shown to be electrophoretically homogeneous and, in rabbits heterozygous at the b locus, to express either one or the other allele. Thus, the antibody released in single microcultures was indeed the product of single B-cell clones and allelic exclusion, once achieved, was maintained throughout clonal proliferation. In the response to streptococcal polysaccharides, analysis of the clonotypes triggered in vitro provided information on mechanisms of clonal dominance. A two-stage culture system was established, where rabbit PBL were precultured at low cell density with antigen before being partitioned in limiting dilution cultures. This method provided a new tool for studies of various cellular aspects of the immune response. Moreover, it allowed the application of the limiting dilution analysis to PBL from unprimed animals. Later, the method was extended with success to human PBL, leading to studies of regulatory aspects of immunity in this species.

Suppressor cells in rabbit peripheral blood.

Rabbit peripheral blood leukocytes (PBL) added to cultures of autologous spleen cells, primed in vivo to sheep red cells, are able to suppress with high efficacy the secondary in vitro response of the spleen cells to that antigen. Removal of nylon wool adherent cells from PBL abolishes the suppressive effect. When the PBL are fractionated by velocity sedimentation the suppressor cells can be separated from the responding cells. The circulating lymphocytes, freed from the inhibiting effect, either by nylon wool absorption or by velocity sedimentation fractionation, are able to give a strong secondary in vitro anti-SRC response, in which the long latency period, usually observed when PBL are stimulated with antigen in culture, is abolished or at least reduced. The suppressor effect present in the PBL is not due to granulocytes, platelets or erythrocytes.

Antigen-specific antibody response induced in cultures of human blood lymphocytes in presence of polyethylene glycol.

Human peripheral blood lymphocytes respond in vitro to sheep red blood cells (SRBC) with the appearance of numerous specific plaque-forming cells, if the Epstein-Barr virus (EBV) is added to the cultures together with the antigen. However, it was found that when polyethylene glycol (mol. wt. 6000) is included in the culture medium at a final concentration of 4%, antigen alone is able to induce an anti-SRBC response, with clear hemolytic plaques of regular size. Addition of EBV causes a further increase in the antigen-specific antibody response.

Expression of antibody-forming cell clones in two-stage cultures: an attempt to separate proliferation and maturation events.

Peripheral blood lymphocytes (PBL) from non-immunized rabbits or from rabbits immunized several months previously with a single dose of sheep red blood cells (SRBC) were cultured at several different cell densities in the presence of antigen for 5 days. The cells were then distributed in 10 microliter microcultures at different cell densities to estimate the frequency of responding units. It was found that a shift up in cell concentration from first to second stage allows a more efficient expression of antibody-forming cell clones. We conclude that, during the first stage at low cell density, the precursor cells proliferate and, when they are partitioned in microcultures and the cell concentration is raised, maturation to antibody-forming cells occurs. This approach allows a separation of the proliferation event from the maturation event and it may prove to be a useful tool in the study of B-cell differentiation.

Long term antibody production in cultures of peripheral blood lymphocytes.

Antigen stimulated cultures of rabbit and human peripheral blood lymphocytes (PBL) were maintained in active antibody synthesis for at least a month. When rabbit PBL were used, the response to a particulate antigen (SRC) was not affected by a change of medium and/or by disruption of the cell to cell contact during culture. On the other hand, the response to a soluble antigen (OA) was markedly increased by changing the medium after the onset of the response. When human PBL were used, any handling that caused disruption of cellular contacts was detrimental for continuation of the specific response. In both the rabbit and the human system the overall cell number did not increase during culture, but an enrichment in the specific cells occurred. These cultures represent a powerful tool both for studies of late events of the immune response and for the aim of establishing antibody producing cell lines.

Interleukin-12 up-regulates the induction of an antigen-specific antibody response in cultures of human lymphocytes.

The influence of interleukin-12 (IL-12) on the induction of a specific antibody response to the T-dependent antigen sheep erythrocytes (SRBC) in cultures of human blood lymphocytes was investigated. The response, evaluated as number of antigen-induced antibody-producing cells, was greatly increased in the presence of IL-12. When a two-stage limiting dilution culture system was used, the plot of the number of seeded cells versus the logarithm of the fraction of negative cultures deviated from linearity in antigen- and IL-12-stimulated cultures. However, linearity was reached when IL-2 was added in the second stage. Under these latter conditions, since single-hit criteria were fulfilled, it was possible to estimate the frequency of SRBC-specific B cell precursors able to respond to the antigen and to show that such frequency was increased upon addition of IL-12. Thus, the enhancing effect of IL-12 may be based on an increased frequency of responding precursor cells. The results here presented demonstrate, to our knowledge for the first time, a definite role of IL-12 in the induction of a specific antibody response in human cells. Further, they stress the importance for such studies of appropriate in vitro systems. Finally, they show that the induction of primary immune responses in cultures of human peripheral blood lymphocytes mostly depends on the proper cytokine balance at different time points.

Possible role of the plasminogen receptor as a site of interaction of the human immunodeficiency virus p24 immunosuppressive heptapeptide Ch7 with the host immune system.

Previous work from our laboratory demonstrated that a synthetic heptapeptide (Ch7: RGSDIAG), corresponding to a conserved sequence of human immunodeficiency virus (HIV) core protein p24 (amino acids 232- 238), was able to specifically abrogate antigen-induced responses in cultures of normal human peripheral blood mononuclear cells (PBMC), probably acting at the level of monocytes. The Ch7 peptide displays sequence homology to human plasminogen. In the present report we show that a compound (6-aminoexanoic acid), known to prevent plasminogen binding to monocyte-like cells, greatly reduced the immunosuppressive capacity of Ch7. We suggest that the plasminogen receptor may represent a target structure on human monocytes for the immunosuppressive p24 sequence.

Interferon-gamma (IFN-gamma) can counteract the in vitro inhibitory effect of an HIV p24 immunosuppressive heptapeptide.

Previous work from our laboratory demonstrated that a synthetic heptapeptide (Ch7), corresponding to a conserved sequence of HIV core protein p24 (aa 232-238), was able to specifically abrogate antigen-induced responses in cultures of normal human peripheral blood lymphocytes (PBL). In the present study we show that Ch7 did not inhibit the induction of IFN-gamma-secreting cells nor the accumulation of IFN-gamma mRNA in antigen-stimulated cultures. However, delayed addition of recombinant human IFN-gamma to Ch7-suppressed cultures was able to restore fully the capacity to mount an antigen-specific antibody response. Thus, although the Ch7 immunosuppressive effect may not be directly related to a decreased production of IFN-gamma, an increased level of this cytokine is certainly able to counteract the negative effect of the peptide.