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This paper summarizes studies on antibody formation in the bone marrow and the suppressive effects of intravenous immunization with allogeneic blood cells on T-cell function in mice. The latter studies were extended by employing the limiting dilution culture system developed in Ivan Lefkovits' laboratory and implemented in collaboration with Lucien Aarden. Thereby, the functional data were complemented with frequencies of alloantigen-activated helper (Th) and suppressor T cells after intravenous alloimmunization. These results led the Rotterdam group to studies on the prevention of rejection of the foetal 'allograft'. Th cells are central in foetal allograft rejection and pregnancy success. Characteristic for human pregnancy is the production of the glycoprotein chorionic gonadotropin (hCG) hormone. The in vivo liberated peptide fragments originating from nicking of the sequence MTRVLQGVLPALPQ in the beta-chain of hCG were considered for their immunoregulating capacity related to pregnancy success. These peptides - prepared synthetically - (MTR, MTRV, LQG, LQGV, VLPALP and others) indeed showed a remarkable spectrum of biological effects (e.g. modulation of angiogenesis, inhibition of septic shock syndrome, prevention of diabetes and reduction of ischaemia-reperfusion damage). The paper interprets and generalizes these findings and projects them into various research directions, especially towards the proteomics framework studies built up in Ivan Lefkovits' laboratory in the nineties. During the time period, when Ivan spent a mini-sabbatical in Rotterdam (months after closing down the BII) more detailed discussions were intiated. This paper is meant to keep the discussions between the involved research groups going on.

SETTING THE STAGEIn a paper in this issue of Scandinavian Journal ofImmunology, Sinclair addresses the role of Fc-signalling inthe modulation of immune responses by passive antibody [1].This is a broad field, encompassing aspects of immuneresponses from autoimmune disease models to cell signallingin vitro. A substantial part of the paper deals in a critical waywith recent results from our laboratory concerning the abilityof immunoglobulin (Ig)G to feedback-regulate in vivo anti-body responses in mice [2,3] and we will limit our reply tothis area. In a field as complicated as antibody feedbackregulation, it is important to define exactly what is discussed.Antibodies of many classes have the capacity to modulateantibody responses, either positively or negatively [4]. IgGhas a dual role, suppressing the response to particulateantigens such as erythrocytes [5–9], and enhancing theresponse to soluble antigens such as keyhole limpethemocyanine (KLH), bovine serum albumin (BSA), andovalbumin (OVA) [3,9–11]. The same monoclonal IgG anti-trinitriphenyl (anti-TNP) can suppress or enhance, dependingon whether TNP is conjugated to an erythrocyte or a solubleantigen [9,11]. Considering the strikingly different structure ofthese two types of immune complexes this is not surprising.While an IgG molecule is much smaller than an erythrocyte(10 nm versus 7 mm in diameter), IgG is on average twice aslarge as BSA. When discussing IgG-mediated immunoregula-tion, it is therefore vital to keep apart studies using particulateversus soluble antigens. Major differences between in vitroand in vivo systems for studying antibody responses exist(e.g., lack of lympoid organ structure in vitro) and unlessspecifically stated, only in vivo systems will be consideredhere.NO EVIDENCE FOR A ROLE OF FCgRIIB INSUPPRESSION OF IN VIVO ANTIBODYRESPONSES TO SRBC BY PASSIVELYADMINISTERED IgGWhy IgG has the capacity to completely inhibit a response toSRBC is a question that has been debated extensively over theyears. Hypotheses put forward are: (i) epitope masking by IgG,preventing recognition of the antigen by B cells, (ii) effectiveelimination of IgG-coated antigen via Fc-dependent phago-cytosis, and (iii) central inhibition of B-cell activation viacocrosslinking of the B-cell receptor and the inhibitoryFcgRIIB. Sinclair argues that the mechanism behind the IgG-mediated suppression of erythrocyte-specific responses wasresolved 30 years ago and that his so called TripartiteInactivation Model ((iii) see above) explains the phenomenon[12]. Some textbooks and reviews also state for a fact that themechanism behind the IgG-mediated suppression has beenproven to be FcgRIIB-mediated negative signalling to B cells.We do not share this opinion. Although this receptorundoubtedly plays an inhibitory role in many experimentalsystems, there are many observations calling for scepticismabout its role in inhibition of erythrocyte responses by passivelyadministered IgG. Firstly, data regarding the Fc-dependence ofsuppression are conflicting. The efficiency of F(ab

A hypothesis is postulated and experimental findings are given that a special kind of cell heterogeneity exists that renders cells differentially sensitive to the epitopal stimulus with the consequence that only a portion of ligand-specific cells engages in the response. The differential sensitivity is based on epigenetically driven cell-to-cell variations in the expressed gene products, such that every cell becomes unique in its molecular profile, and will preserve its individuality in 'sensing' the boundary conditions of the response milieu. The readiness of cells to engage in the response will be termed alacrity. High-alacrity cells are ready to respond under given conditions because their molecular expression pattern - both in qualitative and in quantitative terms - matches the response milieu. This heterogeneity has little to do with the BCR and TCR specificity, that is, not all antigen-specific cells respond to a stimulus, and cells failing to respond do so because their overall molecular pattern is inadequate to the conditions of the response milieu. The corollary to this proposition is that whatever physiological conditions prevail, some ligand-specific cells will likely be ready to engage in the response, because their uniqueness makes them differentially reactive to external signals. Although the pool of cells available for any response is restricted under any given boundary condition, some idle cells are saved to be 'in reserve'. Experiments are described that are compatible with this proposition, and approaches are suggested to elucidate the mechanism of developing and maintaining alacrity. This paper is a contribution to the Centennial conference in honour of Niels Kaj Jerne, held in Lisbon November 2011.

Any discussion of the regulation of effector class must include feedback control on the magnitude of the response. The induced effector activities are biodestructive and ridding. They are, in and of themselves, unspecific with respect to self and nonself. Consequently all responses include some level of innocent bystander pathology. The regulation of magnitude is essential to keeping this level of pathology at an evolutionarily acceptable level. This is my postulated role of suppressor T-cells, more popularly referred to as Treg. In order to perform this function they must be somatically selected to be anti-nonself like all other T/B-cells. If correct, any role that they might play in determining normal tolerance is excluded. As the commentary of Ellestad illustrates, their function to regulate autoimmunity is the consensus view today and, therefore, this competing concept and my analysis of suggested papers should invite a wide ranging debate.

The clinical manifestations of human Chagas disease are associated with distinct and complex host-parasite interactions that directly involve the host's immune system. In this study, we analysed the relationship between the production of intracytoplasmic cytokines after in vitro stimulation with the recombinant antigens CRA (cytoplasmatic repetitive antigen) or FRA (flagellar repetitive antigen) from Trypanosoma cruzi and the chronic cardiac or indeterminate clinical forms of Chagas disease. The chagasic patient groups consisted of 39 individuals, selected at the Chagas Disease Unit of the Oswaldo Cruz University Hospital, whom presented either a cardiac form without cardiac dilatation (CARD 1), cardiac form with cardiac dilatation (CARD 2) or indeterminate form (IND). Blood samples were obtained from these patients and cultured in the presence of CRA or FRA. The cytokines produced by lymphocytes and monocytes after antigen stimulation were analysed by flow cytometry. Our results showed that the IFN-γ and TNF-α, produced by CD8+ T lymphocytes after in vitro stimulation with CRA, differed among chagasic patients with CARD 1, CARD 2 or IND. We propose that these cytokines could be utilized as immunological markers for clinical cardiac forms of Chagas disease. In a prospective study of patients presenting IND and CARD 1, the assay performed in this paper could serve as a tool to monitor therapeutic interventions, thus improving the patient's quality of life.

Passive antibody can both suppress and augment immune responses. Until recently, there was virtual unanimity on the importance of the interaction of the Fc portion of modulating antibody with Fc-receptors (Fc-signalling), especially in experiments involving the suppression by antibody. Experiments reported in the last few years, that do not demonstrate the range of Fc-portion/Fc-receptor influences on the suppression of immune responses by passive antibody, have introduced new uncertainty into this field. The purpose of this paper is to review how the initial controversy on the influence of Fc-signalling in inhibition by passive antibody was resolved. Old and new approaches are suggested that may help in resolving the current uncertainty engendered by recent experimental results that were interpreted to mean that passive suppressive antibody does not utilize the inhibitory FcγRIIB receptor. An understanding of the factors that influence negative Fc-signalling is needed in order to optimize clinical therapies whose action depends on the suppressive property of antibody.

Tuberculosis (TB) is the largest single infectious cause of human mortality. The incidence of TB has remained high in most of the developing world and the disease has recently re-emerged as a public health problem in industrialized countries. The development of a new improved TB vaccine is a highly prioritized international research area, which has been further stimulated by the appearance of multi-drug resistant strains of Mycobacterium tuberculosis. The present status of the attempts to characterize the protective immune response to TB will be reviewed with special emphasis on recent progress in the identification and characterization of target molecules recognized by protective cells. This paper will focus on proteins released from live bacteria and discuss their role in the host-pathogen interaction and the ongoing attempts to use these molecules in TB subunit vaccines.

The mechanisms associated with the modulation of immune response in the chronic phase of human schistosomiasis mansoni infection are complex and involve many cell types. In the present paper the authors demonstrate that antigenic stimulation of peripheral blood mononuclear cells (PBMC) from chronic-intestinal schistosomiasis mansoni patients with polyacrylamide beads (PB) conjugated to Schistosoma mansoni soluble egg antigens (PB-SEA) or adult worm antigen preparation (PB-SWAP) were able to induce a statistically significant increase on the in vitro multinucleated giant cell (MGC) formation after the 15th day in culture. A correlation between an increase in the number of MGC and a decrease in in vitro granuloma formation index to PB-SEA and PB-SWAP was observed. Moreover, the authors demonstrated a down-regulation of lymphocyte proliferative responses to S. mansoni antigens, during the differentiation pathway of monocytes towards MGC formation, due to a decrease in the antigen-presenting capacity of these cells. These phenomena also correlate with a concomitant decrease in the expression of HLA-DR and CD54 adhesion molecules on the surface of MGC. The results suggest that differentiation of monocytes to MGC may be one of the immunoregulatory mechanisms involved in the down-regulation of the granuloma reaction against S. mansoni eggs.

Cellular immune responses are important in the recovery from human cytomegalovirus (HCMV) infection. However, little is known about the CD4+ T cell response and the target antigens (Ag) recognized. In this paper, we have analysed the proliferative T cell response of healthy HCMV seropositive (HCMV+) blood donors to recombinant immediate-early proteins expressed in transfected astrocytoma cells and to total HCMV Ags expressed in infected astrocytoma cells. We found that CD4+ T cells were the major cell population that proliferated in the presence of IE or total HCMV Ags. Among healthy HCMV seropositive blood donors with anti-HCMV specific proliferative response, 33-44% also responded to IE Ags. Moreover, in high responders, the precursor frequencies of cells which proliferated in the presence of total HCMV, IE, or IE1 Ags were high (1/103 to 1/255, 1/2785 to 1/7744 and 1/5190 to 1/13531, respectively). In some donors, the anti-IE response was variable over time, whereas the anti-total HCMV Ags response remained constant, which suggests regulation of the anti-IE response in immunocompetent subjects. Our results suggest that the CD4+ anti-IE1 response represents a significant part of the anti-HCMV proliferative response, both at the population level, and within individual immune systems.

The authors have a long standing interest in immune regulations which control the absence of rejection of a semi-allogeneic fetus by the mother. A previous work described a soluble 40 kDa factor extracted from mouse placenta and capable of inhibiting secondary immune responses in vivo. The present paper reports the following on its mode of action in vivo: (1) it is active even in a fully allogeneic host; (2) it can be administered i. v. or i. p. along with antigen; and (3) the injections of factor and antigen must not be more than 2 days apart for maximum efficacy. Moreover, the results of the study described here indicate also that this factor is a concanavalin A-binding giycoprotein, sensitive to heat and pronase, and different from interleukin 10 (IL-10). Thus, this placental factor appears to be different from previously described immune regulators such as IL-10 and could contribute significantly to immune regulations at the level of the placenta.

Summary This paper addresses the need for receptors involved in recirculation of T lymphocytes both during development and during an immune response or inflammation. Some known receptors present on T lymphocytes which are important in maintaining normal T-cell localization and function, i.e. the integrins, LECAMs, LPAM-1 and H-CAM (CD44) are considered in terms of the specificity which they confer on lymphocyte recirculation. A clear understanding of lymphocyte trafficking patterns and of the receptors involved may provide, for example, novel therapies for treatment of malignant cancer, alleviation of damage caused by lymphocytes during inflammatory responses, and targeting of cells to sites of infection.

Cholera toxin (CT) is a potent stimulator of IgA responses when administered orally and has been shown to promote IgA responses to a second protein such as keyhole limpet haemocyanin (KLH) if this is fed simultaneously. In this paper we show that whilst feeding 5 mg KLH with either 0.5 micrograms CT or 10 micrograms B subunit fails to stimulate a mucosal IgA response to KLH, feeding 0.5 microgram CT and 10 micrograms B subunit together with 5 mg KLH produces a local IgA anti-KLH response as great as that produced by 10 micrograms of whole CT. In addition to stimulating IgA responses in the lamina propria, preliminary results indicate that cellular responses are also stimulated, as we have demonstrated KLH antigen-driven proliferation of cells isolated from groups of mice fed either 10 micrograms CT + 5 mg KLH or 0.5 micrograms CT + 10 micrograms CTB + 5 mg KLH but not mice fed KLH alone or with either 10 micrograms CTB or 0.5 micrograms CT. These results indicate that the mucosal adjuvant action of CT is due to a synergistic effect involving both the GM1 binding of the B subunit and adenylate cyclase activation by the A subunit.

Highly poly morphic age-dependent differences in progression of T and B cells have been found in various strains of inbred mice. In the immune system of C57BL/6 mice these occur first in B cells and, later in life, in T cells. In this paper, we have examined the progression in the functional capacity of C57BL/6 dendritic cells (DC). Age-dependent changes were found in the syngeneic mixed leucocyte reaction, in which the stimulatory capacity of DC increased with age. This increase was independent of the age of the donor of the serum that was added to the culture, and occurred when young donors provided the T cells. Age-dependent changes in DC properties were not detected in terms of direct plaque-forming response, response to con-canavalin A, or in the allogeneic mixed leucocyte reaction.

An assay, the paper radioallergosorbent test (PRAST), for the measurement of specific serum IgE antibody in the rat is described in detail. This assay has been used, in conjunction with a modified PRAST for the determination of relative specific serum IgG antibody and the PRIST assay for total serum IgE [13], to measure specific IgE and IgG and total IgE immune responses in normal parasite infected rats immunized using various protocols. The results indicate that there is a relationship between the basic IgE level and the immune response, i.e. a rat strain with a low constitutive IgE level demonstrates a weak response whereas a high level strain reacts strongly. When PRAST and passive cutaneous anaphylaxis (PCA) were compared, using standardized IgE antibody containing sera, the results were in good agreement. However, PRAST is the preferable assay as it shows less intrinsic variation, is more sensitive than PCA, and is not influenced by high serum IgE levels in the recipient animal.

Immune responsiveness to the streptococcal group polysaccharides at the clonal level can conveniently be monitored by analytical isoelectric focusing combined with autoradiography. The parameters investigated in this paper can be summarized as follows: [1] Within the first week after injection antistreptococcal p0lysaccharide responses are characterized by IgM antibodies, followed by subclass restriction in Balb/c mice to IgG2a antibodies. [2] The clonal pattern of specific IgG2a antibodies established in the second week after immunization does not change within the following 6 weeks. [3] Persistence of a specific response pattern established within a primary immunization course holds for Balb/c mice and rabbits for as long as 12 to 13 months. [4] Repeated immunization courses in mice and rabbits maintain established clonal antibody patterns in toto. Temporal variations of clonal expression are generally quantitative rather than qualitative. B-cell memory within this system is therefore stable and long-lived.